Enhanced reconstitution and autoreconstitution of the hematopoietic compartment

ABSTRACT

The present disclosure relates to the acceleration of hematopoietic compartment reconstitution in a subject in need of hematopoietic stem cell transplantation by administering a composition having a protein transduction domain-MYC (PTD-MYC) fusion protein in combination with hematopoietic stem cell transplantation and to the enhancement of hematopoietic compartment autoreconstitution in a subject in need thereof by administering a composition having a protein transduction domain-MYC (PTD-MYC) fusion protein.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/674,224, filed Jul. 20, 2012, and U.S. Provisional Application No.61/785,691, filed Mar. 14, 2013, both of which are hereby incorporatedby reference in their entirety.

SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 691772000840SeqList.txt,date recorded: Jul. 19, 2013, size: 9 KB).

FIELD

The present disclosure generally relates to hematopoietic compartmentcell formation, cell survival, and cell proliferation by hematopoieticstem cells. In particular, the present disclosure relates to theacceleration of hematopoietic compartment reconstitution and to theenhancement of hematopoietic compartment autoreconstitution.

BACKGROUND

The use of hematopoietic stem cells for bone marrow transplantation hasrevolutionized the approaches used to treat a large number ofhematological malignancies (e.g., leukemias), as well as severalwidespread autoimmune diseases, and is a critical treatment forimmunodeficiencies (Buckley, R H, Annu Rev Immunol 22, 625-655, 2004).The use of hematopoietic stem cell transplantations has also beensuccessful in mitigating the effects of exposure to high levels ofradiation in several instances (Bishop, M R, Stem Cells 15 Suppl 2,305-310, 1997). In addition, hematopoietic stem cell transplantationshave been used to enable the administration of high doses of cytotoxicchemotherapeutic agents to patients who suffer from a number of solidorgan tumors, thus enabling the repopulation of the bone marrowfollowing drug-induced toxicity (Crivellari, G et al., Oncologist 12,79-89, 2007). The use of hematopoietic stem cell transplantation toimprove the rate of engraftment of solid organ transplantations isanother recent application of this medical procedure (Delis, S et al.,Pancreas 32, 1-8, 2006). Recent studies also indicate that bone marrowtransplantation may have value in the treatment of heart disease(Engelmann, M G et al., Curr Opin Mol Ther 8, 396-414, 2006). Althoughthe basis of this effect is unknown, these findings raise thepossibility that hematopoietic stem cells may be reprogrammed to giverise to other tissues (Kiel, M J et al., Dev Biol 283, 29-39, 2005).Accordingly, adult hematopoietic stem cells may have a much broaderutility than, and may provide an alternative to, controversial embryonicstem cell therapy. These therapeutic applications of hematopoietic stemcell transplantation demonstrate the medical and economic impact ofimproving hematopoietic stem cell transplantation.

Several problems have limited the therapeutic application ofhematopoietic stem cell (HSC) transplantation. For example, one majorproblem is the low number of HSCs available for transplants. Patientswho suffer from bone marrow failure, autoimmune diseases, congenitalimmunodeficiencies, or hematological malignancies do not provide a goodsource of HSCs for autologous transplantation (Linker, C, Best Pract ResClin Haematol 20, 77-84, 2007). In addition, some bone marrow failurepatients and cancer patients require multiple rounds of HSCtransplantation in order to achieve full HSC engraftment followingradiation or chemotherapy treatment (Oliansky, D M et al., Biol BloodMarrow Transplant 13, 1-25, 2007). In cases where autologous HSCtransplantation is not possible, there is the additional problem ofidentifying an appropriately histocompatible bone marrow donor. This isgenerally accomplished using registries that have enrolled more than 6million potential donors (de Mello, A N et al., Telemed. Telecare 12Supp13, 64-6, 2006). Additionally, once selected, the donor must undergoa grueling and painful process to mobilize HSCs into the blood followedby 4-5 days of leukapheresis to isolate rare long-term HSCs (Nervi, B etal., J Cell Biochem 99, 690-705, 2006). There have been a number ofnovel approaches aimed at solving the problem of low numbers of HSCsavailable for transplants, by expanding HSCs ex vivo after isolation,but the ability to generate large numbers of long-term repopulating HSCsthat remain available and bioactive over a period of years has remainedelusive (Hoffman, R, Curr Opin Hematol 6, 184-91, 1999).

Another problem that limits the therapeutic application of HSCtransplantation is the time required for the transplanted HSCs toreconstitute the functional and mature hematopoietic lineages (i.e., thehematopoietic compartment) after the ablation of the transplantrecipient's resident immune system. One of the elements required topromote the successful engraftment of transplanted HSCs is the removalof the resident immune system. This is routinely done by total bodyirradiation or chemical ablation. The end result of this process is analmost completely immunocompromised patient that is highly susceptibleto opportunistic infection by environmental microorganisms that humansroutinely interact with during normal activities, such as breathing andeating. This problem is of greater concern with the significant increasein the variety and heterogeneity of iatrogenic infectious agents, manyof which are highly resistant to existing antibiotics. The time requiredfor recovery of mature hematopoietic lineages after an HSC transplantsignificantly affects the risk of the transplant recipient developingand ultimately succumbing to opportunistic infections.

The time required to repopulate mature hematopoietic lineages in an HSCtransplant recipient is affected by several variables. One such variableis the differing recovery times required to repopulate the differenthematopoietic lineages following an HSC transplant. For example, themyeloid compartment, which is composed of monocytes, neutrophils, andbasophils, usually requires 4-8 weeks to recover following HSCtransplantation. The lymphoid compartment requires a significantlylonger recovery time in humans. For example, T-cells, NK cells, andNKT-cells require between 4-8 months to recover, while B-cells requireover 12 months to recover in most individuals. The recovery time of themyeloid lineage cells is critical for the minimal required defensesagainst food-borne and environmental microorganisms. Another variableaffecting the time required to repopulate mature hematopoietic lineagesin an HSC transplant recipient is the number of HSCs available fortransplantation and the size/weight of the recipient. A further variableis the nature of other treatments that an HSC transplant patient mayhave been subjected to prior to the transplantation. In most patientswith some form of cancer, the patient will have been given severalrounds of chemotherapy prior to receiving an HSC transplant. The use ofsuch cytotoxic drugs can impact the bone marrow niches to which thetransplanted HSCs home to and begin the differentiation process. In someinstances where the niches have been destroyed by cytotoxic drugs,several HSC transplants may be required to initially reconstitute theniches and subsequently seed the niches with pluripotent HSCs.

Moreover, the recovery time of mature hematopoietic lineages followingHSC transplantation is largely dependent of the ability of the donorHSCs to find their way to bone marrow niches. Once the HSCs arrive atthe bone marrow niches, they need to establish a molecular crosstalkwith the niche-resident cells. This cross talk is thought to regulatethe nature and levels of cell-intrinsic signals within the HSCs thatregulate their survival, proliferation, self-renewal, anddifferentiation. Thus, failure of HSCs to find their way to bone marrowniches, home properly, or correctly establish such molecular crosstalkwith the niches can result in HSC engraftment failure. Additionally,failure of HSCs to find their way to bone marrow niches can also resultin only a short term recovery of mature hematopoietic lineages or only apartial reconstitution of mature hematopoietic lineages that will slowlysubside as a result of long-term bone marrow failure.

The lag time between ablation of a patient's resident immune system andhematopoietic lineage reconstitution by HSC transplantation is thus oneof the major risk factors for the development of potentially fatalcomplications, such as opportunistic infections or HSC engraftmentfailure. Several approaches have been attempted to decrease the timerequired to reconstitute mature hematopoietic lineages following HSCtransplantation. Examples of such approaches include the use of highernumbers of HSCs for transplantation, partial resident immune systemablation and multiple transplants of smaller numbers of HSCs,pre-conditioning of donor bone marrow to retain its resident T-cells,growth factor treatment of the patient following HSC transplantation,and the use of monoclonal antibodies and/or small molecule modifiersthat target enzymes affecting E-selectin expression in order to improveHSC homing to bone marrow niches after HSC transplantation (Adams G Band Scadden D T, Gene Ther 15, 96-99, 2008; Campbell T B and Broxmeyer HE, Front. Biosc. 13, 1795-1805, 2008; Rocha V and Boxmeyer H, Biology ofBone marrow transplantation 16, S126-S132, 2009; and Hogatt J. et al.,Blood 113, 5444-55, 2009). However, these solutions only provide amodest improvement over current approaches without significantlyaffecting the frequency of fatal opportunistic infections in the patientpopulation.

BRIEF SUMMARY

Accordingly, there is a need for improved approaches for acceleratinghematopoietic compartment reconstitution in a hematopoietic stem cell(HSC) transplant recipient that result in one or more of the following:increase the number of HSCs available for transplantation, increase thenumber of HSCs that productively home to bone marrow niches, reduce therisk of opportunistic infections, and reduce the risk of HSC engraftmentfailure. There is also a need for improved approaches for enhancinghematopoietic compartment autoreconstitution in a subject in need ofhematopoietic compartment autoreconstitution.

In order to meet the above needs, the present disclosure provides novelmethods of enhancing hematopoietic compartment reconstitution (e.g., HSCengraftment) in a subject by treating a population of HSCs with aMYC-composition, such as a protein transduction domain-MYC (PTD-MYC)fusion protein, a Bcl-2-composition, such as a PTD-Bcl-2 fusion protein,or both, prior to transplanting the HSCs into the subject; and to novelmethods of enhancing hematopoietic compartment cell formation in asubject by administering a composition having a MYC-composition, such asa protein transduction domain-MYC (PTD-MYC) fusion protein, aBcl-2-composition, such as a protein transduction domain-Bcl-2(PTD-Bcl-2) fusion protein, or both. Certain aspects relate to methodsof accelerating hematopoietic compartment reconstitution in ahematopoietic stem cell (HSC) transplant recipient by administering acomposition having a MYC-composition, such as a protein transductiondomain-MYC (PTD-MYC) fusion protein, a Bcl-2-composition, such as aprotein transduction domain-Bcl-2 (PTD-Bcl-2) fusion protein, or both incombination with the HSC transplantation. Advantageously, such novelmethods of accelerating hematopoietic compartment reconstitution mayincrease the number of HSCs that productively home to bone marrow nichesof the transplant recipient and/or may enhance the rate of myeloid andlymphoid compartment reconstitution, thereby reducing the risk ofopportunistic infections in the transplant recipient and reducing therisk of HSC engraftment failure.

Additionally, the present disclosure is based, at least in part, on thesurprising discovery that administering a composition having a fusionprotein containing a MYC polypeptide and a protein transduction domain(PTD), such as the HIV TAT protein transduction domain, (TAT-MYC fusionprotein) after HSC transplantation accelerates the recovery time ofmature hematopoietic lineages in an HSC transplant recipient. Withoutwishing to be bound by theory, it is believed that administering aMYC-composition, such as a PTD-MYC fusion protein to an HSC transplantrecipient increases MYC activity in the hematopoietic compartment of therecipient, resulting in enhanced HSC homing to bone marrow niches in therecipient, and enhanced survival of the HSCs after transplantationresulting in a higher likelihood of HSC homing. Enhanced HSC homingimproves the number of transplanted HSCs that can successfully interactwith the cell matrix at bone marrow niches (i.e., productive homing),improves the number of transplanted HSCs at bone marrow niches thatself-renew and produce the relevant progenitor cell types, and improvesthe kinetics of homing to bone marrow niches, which results in theacceleration of hematopoietic compartment reconstitution. Withoutwishing to be bound by theory, it is also believed that administering aMYC-composition, such as a PTD-MYC fusion protein to an HSC transplantrecipient increases MYC activity in the transplanted HSCs, whichincreases the number of HSCs that self-renew and produce the relevantprogenitor cell types and subtypes for reconstituting the functional andmature hematopoietic lineages (i.e., the hematopoietic compartment).Moreover, treating HSCs to be transplanted with a MYC-composition, suchas a PTD-MYC fusion protein approximately from about 10 minutes to about24 hours prior to transplantation also accelerates the recovery time ofmature hematopoietic lineages in an HSC transplant recipient. In someembodiments, the amount of time required to transduce or otherwiseintroduce the MYC-composition into the HSCs is sufficient to achieve anacceleration in the recovery time of mature hematopoietic lineages in anHSC transplant recipient.

Other aspects of the present disclosure also relate to novel methods ofenhancing hematopoietic compartment autoreconstitution in a subject inneed of hematopoietic compartment autoreconstitution by administering acomposition having a MYC-composition, such as a protein transductiondomain-MYC (PTD-MYC) fusion protein, a Bcl-2-composition, such as aprotein transduction domain-Bcl-2 (PTD-Bcl-2) fusion protein, or both,which induce endogenous HSCs to autoreconstitute the hematopoieticcompartment in the subject. Advantageously, such novel methods ofenhancing hematopoietic compartment autoreconstitution may protectpatients, or facilitate recovery, from insults to the hematopoieticcompartment, such as chemotherapy and radiation therapy. Such novelmethods may also be used to treat bone marrow failure syndromes. Withoutwishing to be bound by theory, it is believed that administering aMYC-composition, such as a PTD-MYC fusion protein, a Bcl-2-composition,such as a PTD-Bcl-2 fusion protein, or both to a subject in need ofhematopoietic compartment autoreconstitution increases MYC activity,Bcl-2 activity, or both in the endogenous HSCs, which increases thenumber of HSCs that self-renew and produce the relevant progenitor celltypes and subtypes for autoreconstituting the functional and maturehematopoietic lineages (i.e., the hematopoietic compartment).Furthermore, without wishing to be bound by theory, it is also believedthat MYC activity also enhances HSC localization to bone marrow niches.

While Refaeli et al. state that the protooncogene MYC can break B celltolerance (Refaeli et al. Proc Nat Acad Sci 102(11):4097-4102, 2005),Refaeli et al. provide no guidance regarding the use of PTD-MYC fusionproteins, or the role of MYC in enhancing the ability of HSCs to formhematopoietic compartment cells. Moreover, while US2007/0116691,US2010/0297763, WO2007/047583, US2010/0047217, and WO2010/011644disclose conditionally immortalized long-term stem cells and methods forpreparing differentiated cells, none of these applications disclose thatMYC enhances the ability of exogenously added HSCs to reconstitute thehematopoietic compartment in an HSC transplant recipient, or the abilityof endogenous HSCs to autoreconstitute the hematopoietic compartment ina subject.

In contrast to Refaeli et al., US2007/0116691, US2010/0297763,WO2007/047583, US2010/0047217, and WO2010/011644, the inventors havesurprisingly shown that administering a MYC-composition, such as aPTD-MYC fusion protein in HSC transplant recipients 24 hours afteraccelerates hematopoietic compartment reconstitution by reducing thetime required for recovery of mature hematopoietic lineages by at least50%. The inventors have also surprisingly shown that administering aMYC-composition, such as a PTD-MYC fusion protein in a subject having areduction in their hematopoietic compartment enhances autoreconstitutionin the subject.

Accordingly, the present disclosure relates to a method of enhancinghematopoietic compartment reconstitution to a subject in need ofhematopoietic stem cell transplantation, by: treating a population ofhematopoietic stem cells with a composition containing aMYC-composition, a composition containing a Bcl-2-composition, or both,for less than about 13 days; and administering to the subject, atherapeutically effective amount of the treated population ofhematopoietic stem cells to reconstitute the hematopoietic compartmentof the subject, wherein hematopoietic compartment reconstitution isenhanced compared to hematopoietic compartment reconstitution in asubject that is administered a population of hematopoietic stem cellsthat were not treated with the composition containing a MYC-composition,the composition containing a Bcl-2-composition, or both. Advantageously,a composition of the present disclosure containing a MYC-composition, aBcl-2 composition, or both may also be administered to the subjectreceiving the pre-treated hematopoietic stem cells to maintain and/orfurther enhance hematopoietic compartment reconstitution in the subject.

In certain embodiments that may be combined with any of the precedingembodiments, the population of hematopoietic stem cells is treated withthe composition containing a MYC-composition, the composition containinga Bcl-2-composition, or both, for less than about 12 days, less thanabout 11 days, less than about 10 days, less than about 9 days, lessthan about 8 days, less than about 7 days, less than about 6 days, lessthan about 5 days, less than about 4 days, less than about 2 days, orless than about 1 day. In certain embodiments that may be combined withany of the preceding embodiments, the population of hematopoietic stemcells is treated with the composition containing a MYC-composition, thecomposition containing a Bcl-2-composition, or both, for less than about24 hours, less than about 23 hours, less than about 22 hours, less thanabout 21 hours, less than about 20 hours, less than about 19 hours, lessthan about 18 hours, less than about 17 hours, less than about 16 hours,less than about 15 hours, less than about 14 hours, less than about 13hours, less than about 12 hours, less than about 11 hours, less thanabout 10 hours, less than about 9 hours, less than about 8 hours, lessthan about 7 hours, less than about 6 hours, less than about 5 hours,less than about 4 hours, less than about 3 hours, less than about 2hours, or less than about 1 hour. In certain embodiments that may becombined with any of the preceding embodiments, the population ofhematopoietic stem cells is treated with the composition containing aMYC-composition, the composition containing a Bcl-2-composition, orboth, for less than about 60 minutes, less than about 55 minutes, lessthan about 50 minutes, less than about 45 minutes, less than about 40minutes, less than about 35 minutes, less than about 30 minutes, lessthan about 29 minutes, less than about 28 minutes, less than about 27minutes, less than about 26 minutes, less than about 25 minutes, lessthan about 24 minutes, less than about 23 minutes, less than about 22minutes, less than about 21 minutes, less than about 20 minutes, lessthan about 19 minutes, less than about 18 minutes, less than about 17minutes, less than about 16 minutes, less than about 15 minutes, lessthan about 14 minutes, less than about 13 minutes, less than about 12minutes, less than about 11 minutes, or less than about 10 minutes. Incertain embodiments that may be combined with any of the precedingembodiments, the population of hematopoietic stem cells is treated withthe composition containing a MYC-composition. In certain embodimentsthat may be combined with any of the preceding embodiments, thepopulation of hematopoietic stem cells is treated with the compositioncontaining a Bcl-2-composition. In certain embodiments that may becombined with any of the preceding embodiments, the population ofhematopoietic stem cells is treated with the composition containing aMYC-composition and the composition containing a Bcl-2-composition. Incertain embodiments that may be combined with any of the precedingembodiments, the therapeutically effective amount of the compositioncontaining a MYC-composition is at least 0.5 μ/ml, at least 0.6 μ/ml, atleast 0.7 μml, at least 0.8 μ/ml, at least 0.9 μ/ml, at least 1 μ/ml, atleast 2 μ/ml, at least 3 μ/ml, at least 4 μ/ml, at least 5 μ/ml, atleast 6 μ/ml, at least 7 μ/ml, at least 8 μ/ml, at least 9 μ/ml, atleast 10 μ/ml, at least 15 μ/ml, at least 20 μ/ml, at least 25 μ/ml, atleast 30 μ/ml, at least 35 μ/ml, at least 40 μ/ml, at least 45 μ/ml, atleast 50 μ/ml, at least 55 μ/ml, at least 60 μ/ml, at least 65 μ/ml, atleast 70 μ/ml, at least 75 μ/ml, at least 80 μ/ml, at least 85 μ/ml, atleast 90 μ/ml, at least 95 μ/ml, or at least 100 μ/ml. In certainembodiments that may be combined with any of the preceding embodiments,the Bcl-2-composition contains a Bcl-2 polypeptide, a homologue thereof,an analogue thereof, or a biologically active fragment thereof. Incertain embodiments that may be combined with any of the precedingembodiments, the Bcl-2-composition contains a protein transductiondomain (PTD). In certain embodiments that may be combined with any ofthe preceding embodiments, the Bcl-2-composition is a PTD-Bcl-2 fusionprotein. In certain embodiments that may be combined with any of thepreceding embodiments, the Bcl-2-composition is a TAT-Bcl-2 fusionprotein. In certain embodiments that may be combined with any of thepreceding embodiments, the therapeutically effective amount of thecomposition containing a Bcl-2-composition is at least 0.5 μ/ml, atleast 0.6 μ/ml, at least 0.7 μ/ml, at least 0.8 μ/ml, at least 0.9 μ/ml,at least 1 μ/ml, at least 2 μ/ml, at least 3 μ/ml, at least 4 μ/ml, atleast 5 μ/ml, at least 6 μ/ml, at least 7 μ/ml, at least 8 μ/ml, atleast 9 μ/ml, at least 10 μ/ml, at least 15 μ/ml, at least 20 μ/ml, atleast 25 μ/ml, at least 30 μ/ml, at least 35 μ/ml, at least 40 μ/ml, atleast 45 μ/ml, at least 50 μ/ml, at least 55 μ/ml, at least 60 μ/ml, atleast 65 μ/ml, at least 70 μ/ml, at least 75 μ/ml, at least 80 μ/ml, atleast 85 μ/ml, at least 90 μ/ml, at least 95 μ/ml, or at least 100 μ/ml.In certain embodiments that may be combined with any of the precedingembodiments, the composition containing a MYC-composition furthercontains a pharmaceutically acceptable carrier. In certain embodimentsthat may be combined with any of the preceding embodiments, thepopulation of hematopoietic stem cells is washed prior to beingadministered to the subject in need thereof. In certain embodiments thatmay be combined with any of the preceding embodiments, the population ofhematopoietic stem cells is administered to the subject in need thereofwithout washing the population of hematopoietic stem cells. In certainembodiments that may be combined with any of the preceding embodiments,the subject has or had a hematological malignancy, a myeloma, multiplemyeloma, a leukemia, acute lymphoblastic leukemia, chronic lymphocyticleukemia, a lymphoma, indolent lymphoma, non-Hodgkin lymphoma, diffuse Bcell lymphoma, follicular lymphoma, mantle cell lymphoma, T celllymphoma, Hodgkin lymphoma, a neuroblastoma, a retinoblastoma, ShwachmanDiamond syndrome, a brain tumor, Ewing's Sarcoma, a Desmoplastic smallround cell tumor, a relapsed germ cell tumor, a hematological disorder,a hemoglobinopathy, an autoimmune disorder, juvenile idiopathicarthritis, systemic lupus erythematosus, severe combinedimmunodeficiency, congenital neutropenia with defective stem cells,severe aplastic anemia, a sickle-cell disease, a myelodysplasticsyndrome, chronic granulomatous disease, a metabolic disorder, Hurlersyndrome, Gaucher disease, osteopetrosis, malignant infantileosteopetrosis, heart disease, HIV, or AIDS. In certain embodiments thatmay be combined with any of the preceding embodiments, the subject hashad an organ transplant. In certain embodiments that may be combinedwith any of the preceding embodiments, the population of hematopoieticstem cells were obtained from bone marrow, from peripheral blood cells,from peripheral blood cells that have undergone apheresis, fromperipheral blood cells that have undergone leukapheresis, from umbilicalcord blood, from amniotic fluid, from cultured HSC cells, from animmortalized HSC cell line, or from a conditionally immortalized HSCcell line. In certain embodiments that may be combined with any of thepreceding embodiments, the treated population of hematopoietic stemcells is administered as a step in a hematopoietic stem cell (HSC)transplantation procedure. In certain embodiments that may be combinedwith any of the preceding embodiments, the HSC transplantation procedureis a myeloablative HSC transplantation procedure. In certain embodimentsthat may be combined with any of the preceding embodiments, the HSCtransplantation procedure is a non-myeloablative HSC transplantationprocedure. In certain embodiments that may be combined with any of thepreceding embodiments, the HSC transplantation is an autologous HSCtransplantation or an allogenic HSC transplantation. In certainembodiments that may be combined with any of the preceding embodiments,administration of the treated population of hematopoietic stem cellsaccelerates hematopoietic compartment reconstitution after HSCtransplantation in the subject. In certain embodiments that may becombined with any of the preceding embodiments, administering thetreated population of hematopoietic stem cells achieves an at least 50%acceleration in hematopoietic compartment reconstitution, compared tohematopoietic compartment reconstitution in a subject that isadministered a population of hematopoietic stem cells that were nottreated with the composition containing a MYC-composition, thecomposition containing a Bcl-2-composition, or both. In certainembodiments that may be combined with any of the preceding embodiments,the accelerated hematopoietic compartment reconstitution in the subjectresults in T cell compartment reconstitution that is accelerated by atleast 50%, compared to T cell reconstitution in a subject that isadministered a population of hematopoietic stem cells that were nottreated with the composition containing a MYC-composition, thecomposition containing a Bcl-2-composition, or both. In certainembodiments that may be combined with any of the preceding embodiments,the accelerated hematopoietic compartment reconstitution in the subjectresults in B cell compartment reconstitution that is accelerated by atleast 50%, compared to B cell reconstitution in a subject that isadministered a population of hematopoietic stem cells that were nottreated with the composition containing a MYC-composition, thecomposition containing a Bcl-2-composition, or both. In certainembodiments that may be combined with any of the preceding embodiments,the accelerated hematopoietic compartment reconstitution in the subjectresults in NK-cell compartment reconstitution that is accelerated by atleast 50%, compared to NK-cell reconstitution in a subject that isadministered a population of hematopoietic stem cells that were nottreated with the composition containing a MYC-composition, thecomposition containing a Bcl-2-composition, or both. In certainembodiments that may be combined with any of the preceding embodiments,he accelerated hematopoietic compartment reconstitution in the subjectresults in myeloid cell compartment reconstitution that is acceleratedby at least 50%, compared to myeloid cell compartment reconstitution ina subject that is administered a population of hematopoietic stem cellsthat were not treated with the composition containing a MYC-composition,the composition containing a Bcl-2-composition, or both. In certainembodiments that may be combined with any of the preceding embodiments,the accelerated hematopoietic compartment reconstitution in the subjectresults in neutrophil recovery that is accelerated by at least 50%,compared to neutrophil recovery in a subject that is administered apopulation of hematopoietic stem cells that were not treated with thecomposition containing a MYC-composition, the composition containing aBcl-2-composition, or both. In certain embodiments that may be combinedwith any of the preceding embodiments, administration of the compositioncontaining a MYC-composition results in a 50% increase in HSC productivehoming to bone marrow niches in the subject, compared to a subject thatis administered a population of hematopoietic stem cells that were nottreated with the composition containing a MYC-composition, thecomposition containing a Bcl-2-composition, or both. In certainembodiments that may be combined with any of the preceding embodiments,further including administering a third composition containing aMYC-composition, a Bcl-2-composition, or both; and optionally at leastone cytokine, growth factor, antibody, and/or small molecule modifier.In certain embodiments that may be combined with any of the precedingembodiments, the third composition further includes a pharmaceuticallyacceptable carrier. In certain embodiments that may be combined with anyof the preceding embodiments, the population of hematopoietic stem cellsis a population of human hematopoietic stem cells. In certainembodiments that may be combined with any of the preceding embodiments,the MYC-composition contains a MYC polypeptide, a homologue thereof, ananalogue thereof, or a biologically active fragment thereof. In certainembodiments that may be combined with any of the preceding embodiments,the MYC-compound contains a protein transduction domain (PTD). Incertain embodiments that may be combined with any of the precedingembodiments, the MYC-composition is a PTD-MYC fusion protein. In certainembodiments that may be combined with any of the preceding embodiments,the MYC-composition is a TAT-MYC fusion protein. In certain embodimentsthat may be combined with any of the preceding embodiments, the subjectis a human patient. In certain embodiments that may be combined with anyof the preceding embodiments, the subject is a non-human animal.

Other aspects of the present disclosure relate to a method of enhancinghematopoietic compartment cell formation in a subject, by: administeringa therapeutically effective amount of a composition containing aMYC-composition, a Bcl-2-composition, or both to a subject in needthereof, where hematopoietic compartment formation is enhanced comparedto hematopoietic compartment formation in a subject that is notadministered the composition.

In certain embodiments that may be combined with any of the precedingembodiments, the composition contains a MYC-composition. In certainembodiments that may be combined with any of the preceding embodiments,the composition contains a Bcl-2-composition. In certain embodimentsthat may be combined with any of the preceding embodiments, thecomposition contains a MYC-composition and a Bcl-2-composition. Incertain embodiments that may be combined with any of the precedingembodiments, the Bcl-2-composition contains a Bcl-2 polypeptide, ahomologue thereof, an analogue thereof, or a biologically activefragment thereof. In certain embodiments that may be combined with anyof the preceding embodiments, the Bcl-2-composition comprises a proteintransduction domain (PTD). In certain embodiments that may be combinedwith any of the preceding embodiments, the Bcl-2-composition is aPTD-Bcl-2 fusion protein. In certain embodiments that may be combinedwith any of the preceding embodiments, the Bcl-2-composition is aTAT-Bcl-2 fusion protein. In certain embodiments that may be combinedwith any of the preceding embodiments, the MYC-composition contains aMYC polypeptide, a homologue thereof, an analogue thereof, or abiologically active fragment thereof. In certain embodiments that may becombined with any of the preceding embodiments, the MYC-compoundcontains a protein transduction domain (PTD). In certain embodimentsthat may be combined with any of the preceding embodiments, theMYC-composition is a PTD-MYC fusion protein. In certain embodiments thatmay be combined with any of the preceding embodiments, theMYC-composition is a TAT-MYC fusion protein. In certain embodiments thatmay be combined with any of the preceding embodiments, the subject is inneed or was in need of hematopoietic stem cell (HSC) transplantation. Incertain embodiments, the subject has or had a hematological malignancy,a myeloma, multiple myeloma, a leukemia, acute lymphoblastic leukemia,chronic lymphocytic leukemia, a lymphoma, indolent lymphoma, non-Hodgkinlymphoma, diffuse B cell lymphoma, follicular lymphoma, mantle celllymphoma, T cell lymphoma, Hodgkin lymphoma, a neuroblastoma, aretinoblastoma, Shwachman Diamond syndrome, a brain tumor, Ewing'sSarcoma, a Desmoplastic small round cell tumor, a relapsed germ celltumor, a hematological disorder, a hemoglobinopathy, an autoimmunedisorder, juvenile idiopathic arthritis, systemic lupus erythematosus,severe combined immunodeficiency, congenital neutropenia with defectivestem cells, severe aplastic anemia, a sickle-cell disease, amyelodysplastic syndrome, chronic granulomatous disease, a metabolicdisorder, Hurler syndrome, Gaucher disease, osteopetrosis, malignantinfantile osteopetrosis, heart disease, HIV, or AIDS. In certainembodiments, the subject has had an organ transplant. In certainembodiments that may be combined with any of the preceding embodiments,the method further includes administering a therapeutically effectiveamount of a second composition containing HSCs to achieve hematopoieticcompartment reconstitution in the subject. In certain embodiments, thecomposition containing a MYC-composition, a Bcl-2-composition, or both,a Bcl-2-composition, or both is administered before, after, orconcurrently with the administering of the second composition. Incertain embodiments, the composition containing a MYC-composition, aBcl-2-composition, or both, a Bcl-2-composition, or both is administeredat least 5 days, at least 4 days, at least 3 days, at least 2 days, orat least 1 day before the administering of the second composition. Incertain embodiments, the composition containing a MYC-composition, aBcl-2-composition, or both, a Bcl-2-composition, or both is administeredconcurrently with the administering of the second composition. Incertain embodiments, the composition containing a MYC-composition, aBcl-2-composition, or both, a Bcl-2-composition, or both is administeredat least 1 day, at least 2 days, at least three days, at least 4 days,at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, orat least 3 weeks after the administering of the second composition. Incertain embodiments that may be combined with any of the precedingembodiments, administration of the composition containing aMYC-composition, a Bcl-2-composition, or both, a Bcl-2-composition, orboth results in an expansion of the HSCs contained in the secondcomposition. In certain embodiments that may be combined with any of thepreceding embodiments, the second composition containing HSCs wascultured in the presence of a MYC-composition, a Bcl-2-composition, orboth, before the administering of the second composition. In certainembodiments, culturing the second composition in the presence of theMYC-composition, Bcl-2-composition, or both conditionally immortalizedthe HSCs. In certain embodiments, immortalization of the HSCs resultedin an expansion of the HSCs. In certain embodiments that may be combinedwith any of the preceding embodiments, the second composition asadministered includes the MYC-composition, Bcl-2-composition, or both asa result of being cultured in the presence of the MYC-composition,Bcl-2-composition, or both. In certain embodiments that may be combinedwith any of the preceding embodiments, the HSCs were obtained from bonemarrow, from peripheral blood cells, from peripheral blood cells thathave undergone apheresis, from peripheral blood cells that haveundergone leukapheresis, from umbilical cord blood, from amniotic fluid,from cultured HSC cells, from an immortalized HSC cell line, or from aconditionally immortalized HSC cell line. In certain embodiments thatmay be combined with any of the preceding embodiments, the HSCs arepresent in bone marrow, in peripheral blood cells, in peripheral bloodcells that have undergone apheresis, in peripheral blood cells that haveundergone leukapheresis, in umbilical cord blood, or in amniotic fluid.In certain embodiments that may be combined with any of the precedingembodiments, the second composition is administered as a step in an HSCtransplantation procedure. In certain embodiments, the HSCtransplantation procedure is a myeloablative HSC transplantationprocedure. In certain embodiments, the HSC transplantation procedure isa non-myeloablative HSC transplantation procedure. In certainembodiments that may be combined with any of the preceding embodiments,the HSC transplantation is an autologous HSC transplantation or anallogenic HSC transplantation. In certain embodiments that may becombined with any of the preceding embodiments, administration of thecomposition containing a MYC-composition, a Bcl-2-composition, or both,a Bcl-2-composition, or both accelerates hematopoietic compartmentreconstitution after HSC transplantation in the subject. In certainembodiments, administering the composition containing a MYC-composition,a Bcl-2-composition, or both, a Bcl-2-composition, or both achieves anat least 50% acceleration in hematopoietic compartment reconstitutioncompared to hematopoietic compartment reconstitution in a subject thatis not administered the composition. In certain embodiments that may becombined with any of the preceding embodiments, the acceleratedhematopoietic compartment reconstitution in the subject results in Tcell compartment reconstitution that is accelerated by at least 50%compared to T cell reconstitution in a subject that is not administeredthe composition. In certain embodiments that may be combined with any ofthe preceding embodiments, the accelerated hematopoietic compartmentreconstitution in the subject results in B cell compartmentreconstitution that is accelerated by at least 50% compared to B cellreconstitution in a subject that is not administered the composition. Incertain embodiments that may be combined with any of the precedingembodiments, the accelerated hematopoietic compartment reconstitution inthe subject results in NK-cell compartment reconstitution that isaccelerated by at least 50% compared to NK-cell reconstitution in asubject that is not administered the composition. In certain embodimentsthat may be combined with any of the preceding embodiments, theaccelerated hematopoietic compartment reconstitution in the subjectresults in myeloid cell compartment reconstitution that is acceleratedby at least 50% compared to myeloid cell compartment reconstitution in asubject that is not administered the composition. In certain embodimentsthat may be combined with any of the preceding embodiments, theaccelerated hematopoietic compartment reconstitution in the subjectresults in neutrophil recovery that is accelerated by at least 50%compared to neutrophil recovery in a subject that is not administeredthe composition. In certain embodiments that may be combined with any ofthe preceding embodiments, administration of the composition containinga MYC-composition, a Bcl-2-composition, or both, a Bcl-2-composition, orboth results in a 50% increase in HSC productive homing to bone marrowniches in the subject. In certain embodiments that may be combined withany of the preceding embodiments, the therapeutically effective amountof the second composition administered to the subject is less when thecomposition containing a MYC-composition, a Bcl-2-composition, or both,a Bcl-2-composition, or both is administered compared to the amountrequired when the composition containing a MYC-composition, aBcl-2-composition, or both, a Bcl-2-composition, or both is notadministered. In certain embodiments that may be combined with any ofthe preceding embodiments, further including administering a thirdcomposition containing at least one cytokine, growth factor, antibody,and/or small molecule modifier. In certain embodiments, the thirdcomposition further contains a pharmaceutically acceptable carrier. Incertain embodiments that may be combined with any of the precedingembodiments, the HSCs contained in the second composition are humanHSCs. In certain embodiments, the subject is in need or was in need ofhematopoietic compartment autoreconstitution. In certain embodiments,the MYC-composition, Bcl-2-composition, or both induces endogenoushematopoietic stem cells (HSCs) to autoreconstitute the hematopoieticcompartment in the subject. In certain embodiments that may be combinedwith any of the preceding embodiments, the hematopoietic compartmentautoreconstitution is enhanced compared to hematopoietic compartmentautoreconstitution in a subject that is not administered the compositioncontaining a MYC-composition, a Bcl-2-composition, or both, aBcl-2-composition, or both. In certain embodiments that may be combinedwith any of the preceding embodiments, the enhanced hematopoieticcompartment autoreconstitution in the subject results in enhanced T cellcompartment autoreconstitution, enhanced B cell compartmentautoreconstitution, enhanced NK-cell compartment autoreconstitution,enhanced myeloid cell compartment autoreconstitution, or neutrophilrecovery. In certain embodiments that may be combined with any of thepreceding embodiments, administration of the composition containing aMYC-composition, a Bcl-2-composition, or both, a Bcl-2-composition, orboth results in an expansion of endogenous HSCs. In certain embodimentsthat may be combined with any of the preceding embodiments, the subjectis undergoing or has undergone chemotherapy. In certain embodiments,administration of the composition containing a MYC-composition, aBcl-2-composition, or both, a Bcl-2-composition, or both prevents adecrease in hematopoietic compartment cells due to the chemotherapy. Incertain embodiments that may be combined with any of the precedingembodiments, administration of the composition containing aMYC-composition, a Bcl-2-composition, or both prevents a decrease in theamount of endogenous HSCs due to the chemotherapy. In certainembodiments that may be combined with any of the preceding embodiments,the subject is undergoing or has undergone radiation therapy. In certainembodiments, administration of the composition containing aMYC-composition, a Bcl-2-composition, or both prevents a decrease inhematopoietic compartment cells due to the radiation therapy. In certainembodiments that may be combined with any of the preceding embodiments,administration of the composition containing a MYC-composition, aBcl-2-composition, or both prevents a decrease in the amount ofendogenous HSCs due to the radiation therapy. In certain embodimentsthat may be combined with any of the preceding embodiments, the subjecthas a bone marrow failure syndrome. In certain embodiments, the bonemarrow failure syndrome is aplastic anemia or Gulf War syndrome. Incertain embodiments, the bone marrow failure syndrome is an inheritedbone marrow failure syndrome (IBMFS). In certain embodiments, the IBMFSis selected from amegakaryocytic thrombocytopenia, Diamond-Blackfananemia, dyskeratosis congenita, fanconi anemia, Pearson syndrome, severecongenital neutropenia, Shwachman-Diamond syndrome, and thrombocytopeniaabsent radii, IVIC syndrome, WT syndrome, radio-ulnar synostosis, andataxia pancytopenia. In certain embodiments that may be combined withany of the preceding embodiments, administration of the compositioncontaining a MYC-composition, a Bcl-2-composition, or both prevents adecrease in hematopoietic compartment cells due to the bone marrowfailure syndrome. In certain embodiments that may be combined with anyof the preceding embodiments, administration of the compositioncontaining a MYC-composition, a Bcl-2-composition, or both prevents adecrease in the amount of endogenous HSCs due to the bone marrow failuresyndrome. In certain embodiments that may be combined with any of thepreceding embodiments, the therapeutically effective amount of thecomposition containing a MYC-composition is at least 0.1 mg/kg, at least0.2 mg/kg, at least 0.3 mg/kg, at least 0.4 mg/kg, at least 0.5 mg/kg,at least 0.6 mg/kg, at least 0.7 mg/kg, at least 0.8 mg/kg, at least 0.9mg/kg, at least 1 mg/kg, at least 2 mg/kg, at least 3 mg/kg, at least 4mg/kg, at least 5 mg/kg, at least 6 mg/kg, at least 7 mg/kg, at least 8mg/kg, at least 9 mg/kg, at least 10 mg/kg, at least 20 mg/kg, at least30 mg/kg, at least 40 mg/kg, or at least 50 mg/kg of the subject'sweight. In certain embodiments that may be combined with any of thepreceding embodiments, the therapeutically effective amount of thecomposition comprising a Bcl-2-composition is at least 0.1 mg/kg, atleast 0.2 mg/kg, at least 0.3 mg/kg, at least 0.4 mg/kg, at least 0.5mg/kg, at least 0.6 mg/kg, at least 0.7 mg/kg, at least 0.8 mg/kg, atleast 0.9 mg/kg, at least 1 mg/kg, at least 2 mg/kg, at least 3 mg/kg,at least 4 mg/kg, at least 5 mg/kg, at least 6 mg/kg, at least 7 mg/kg,at least 8 mg/kg, at least 9 mg/kg, at least 10 mg/kg, at least 20mg/kg, at least 30 mg/kg, at least 40 mg/kg, or at least 50 mg/kg of thesubject's weight. In certain embodiments that may be combined with anyof the preceding embodiments, the composition containing aMYC-composition, a Bcl-2-composition, or both further contains apharmaceutically acceptable carrier. In certain embodiments that may becombined with any of the preceding embodiments, the subject is a humanpatient. In certain embodiments that may be combined with any of thepreceding embodiments, the subject is a non-human animal.

Other aspects of the present disclosure relate to a method ofaccelerating hematopoietic compartment reconstitution afterhematopoietic stem cell (HSC) transplantation in a subject, by: a)administering a therapeutically effective amount of a first compositioncontaining HSCs to achieve hematopoietic compartment reconstitution in asubject in need thereof; and b) administering a second compositioncontaining a MYC-composition, a Bcl-2-composition, or both to thesubject, where administering the second composition achieves an at least50% acceleration in hematopoietic compartment reconstitution compared tohematopoietic compartment reconstitution in a subject that is notadministered the second composition.

Other aspects of the present disclosure relate to a method of enhancinghematopoietic compartment autoreconstitution in a subject, by:administering a therapeutically effective amount of a compositioncontaining a MYC-composition, a Bcl-2-composition, or both to a subjectin need of hematopoietic compartment autoreconstitution, where theMYC-composition, the Bcl-2-composition, or both induces endogenoushematopoietic stem cells (HSCs) to autoreconstitute the hematopoieticcompartment in the subject, and where hematopoietic compartmentautoreconstitution is enhanced compared to hematopoietic compartmentautoreconstitution in a subject that is not administered thecomposition.

Other aspects of the present disclosure relate to a method of treating adecrease in hematopoietic compartment cells due to chemotherapy, by:administering a therapeutically effective amount of a compositioncontaining a MYC-composition, a Bcl-2-composition, or both to a subjectthat is undergoing or has undergone chemotherapy, where theMYC-composition, Bcl-2-composition, or both induces endogenoushematopoietic stem cells (HSCs) to autoreconstitute the hematopoieticcompartment in the subject.

Other aspects of the present disclosure relate to a method of treating adecrease in hematopoietic compartment cells due to radiation therapy,by: administering a therapeutically effective amount of a compositioncontaining a MYC-composition, a Bcl-2-composition, or both to a subjectthat is undergoing or has undergone radiation therapy, where theMYC-composition, Bcl-2-composition, or both induces endogenoushematopoietic stem cells (HSCs) to autoreconstitute the hematopoieticcompartment in the subject.

Other aspects of the present disclosure relate to a method of treating abone marrow failure syndrome, by: administering a therapeuticallyeffective amount of a composition containing a MYC-composition, aBcl-2-composition, or both to a subject having a bone marrow failuresyndrome, where the MYC-composition, Bcl-2-composition, or both inducesendogenous hematopoietic stem cells (HSCs) to autoreconstitute thehematopoietic compartment in the subject.

Other aspects of the present disclosure relate to a use of a compositioncontaining a MYC-composition, a Bcl-2-composition, or both in a subjectthat is in need or was in need of hematopoietic compartment cellformation to enhance hematopoietic compartment cell formation, where useof the composition enhances hematopoietic compartment formation comparedto hematopoietic compartment formation in a patient that does notreceive the composition.

In certain embodiments that may be combined with any of the precedingembodiments, the subject is in need or was in need of hematopoietic stemcell (HSC) transplantation. In certain embodiments, the subject has orhad a hematological malignancy, a myeloma, multiple myeloma, a leukemia,acute lymphoblastic leukemia, chronic lymphocytic leukemia, a lymphoma,indolent lymphoma, non-Hodgkin lymphoma, diffuse B cell lymphoma,follicular lymphoma, mantle cell lymphoma, T cell lymphoma, Hodgkinlymphoma, a neuroblastoma, a retinoblastoma, Shwachman Diamond syndrome,a brain tumor, Ewing's Sarcoma, a Desmoplastic small round cell tumor, arelapsed germ cell tumor, a hematological disorder, a hemoglobinopathy,an autoimmune disorder, juvenile idiopathic arthritis, systemic lupuserythematosus, severe combined immunodeficiency, congenital neutropeniawith defective stem cells, severe aplastic anemia, a sickle-celldisease, a myelodysplastic syndrome, chronic granulomatous disease, ametabolic disorder, Hurler syndrome, Gaucher disease, osteopetrosis,malignant infantile osteopetrosis, heart disease, HIV, or AIDS. Incertain embodiments, the subject has had an organ transplant. In certainembodiments that may be combined with any of the preceding embodiments,the subject is undergoing or underwent and HSC transplant. In certainembodiments, the composition containing a MYC-composition, aBcl-2-composition, or both is administered before, after, orconcurrently with the HSC transplant. In certain embodiments, thecomposition containing a MYC-composition, a Bcl-2-composition, or bothis administered at least 5 days, at least 4 days, at least 3 days, atleast 2 days, or at least 1 day before HSC transplantation. In certainembodiments, the composition containing a MYC-composition, aBcl-2-composition, or both is administered concurrently with HSCtransplantation. In certain embodiments, the composition containing aMYC-composition, a Bcl-2-composition, or both is administered at least 1day, at least 2 days, at least three days, at least 4 days, at least 5days, at least 6 days, at least 1 week, at least 2 weeks, or at least 3weeks after the HSC transplantation. In certain embodiments that may becombined with any of the preceding embodiments, administration of thecomposition containing a MYC-composition, a Bcl-2-composition, or bothresults in an expansion of the transplanted HSCs. In certain embodimentsthat may be combined with any of the preceding embodiments, thetransplanted HSCs were cultured in the presence of a MYC-composition, aBcl-2-composition, or both prior to HSC transplantation. In certainembodiments, culturing the HSCs in the presence of the MYC-composition,Bcl-2-composition, or both conditionally immortalized the HSCs. Incertain embodiments, immortalization of the HSCs resulted in anexpansion of the HSCs. In certain embodiments that may be combined withany of the preceding embodiments, the transplanted HSCs included theMYC-composition, Bcl-2-composition, or both as a result of beingcultured in the presence of the MYC-composition, Bcl-2-composition, orboth. In certain embodiments that may be combined with any of thepreceding embodiments, the transplanted HSCs were obtained from bonemarrow, from peripheral blood cells, from peripheral blood cells thathave undergone apheresis, from peripheral blood cells that haveundergone leukapheresis, from umbilical cord blood, from amniotic fluid,from cultured HSC cells, from an immortalized HSC cell line, or from aconditionally immortalized HSC cell line. In certain embodiments thatmay be combined with any of the preceding embodiments, the HSCtransplantation procedure is or was a myeloablative HSC transplantationprocedure. In certain embodiments that may be combined with any of thepreceding embodiments, the HSC transplantation procedure is or was anon-myeloablative HSC transplantation procedure. In certain embodimentsthat may be combined with any of the preceding embodiments, the HSCtransplantation is or was an autologous HSC transplantation. In certainembodiments that may be combined with any of the preceding embodiments,the HSC transplantation is or was an allogenic HSC transplantation. Incertain embodiments that may be combined with any of the precedingembodiments, administration of the composition containing aMYC-composition, a Bcl-2-composition, or both accelerates hematopoieticcompartment reconstitution after HSC transplantation in the subject. Incertain embodiments, administering the composition containing aMYC-composition, a Bcl-2-composition, or both achieves an at least 50%acceleration in hematopoietic compartment reconstitution compared tohematopoietic compartment reconstitution in a subject that is notadministered the composition. In certain embodiments that may becombined with any of the preceding embodiments, the acceleratedhematopoietic compartment reconstitution in the subject results in Tcell compartment reconstitution that is accelerated by at least 50%compared to T cell reconstitution in a subject that is not administeredthe composition. In certain embodiments that may be combined with any ofthe preceding embodiments, the accelerated hematopoietic compartmentreconstitution in the subject results in B cell compartmentreconstitution that is accelerated by at least 50% compared to B cellreconstitution in a subject that is not administered the composition. Incertain embodiments that may be combined with any of the precedingembodiments, the accelerated hematopoietic compartment reconstitution inthe subject results in NK-cell compartment reconstitution that isaccelerated by at least 50% compared to NK-cell reconstitution in asubject that is not administered the composition. In certain embodimentsthat may be combined with any of the preceding embodiments, theaccelerated hematopoietic compartment reconstitution in the subjectresults in myeloid cell compartment reconstitution that is acceleratedby at least 50% compared to myeloid cell compartment reconstitution in asubject that is not administered the composition. In certain embodimentsthat may be combined with any of the preceding embodiments, theaccelerated hematopoietic compartment reconstitution in the subjectresults in neutrophil recovery that is accelerated by at least 50%compared to neutrophil recovery in a subject that is not administeredthe composition. In certain embodiments that may be combined with any ofthe preceding embodiments, administration of the composition containinga MYC-composition, a Bcl-2-composition, or both results in a 50%increase in HSC productive homing to bone marrow niches in the subject.In certain embodiments that may be combined with any of the precedingembodiments, further including administering a second compositioncontaining at least one cytokine, growth factor, antibody, and/or smallmolecule modifier. In certain embodiments, the third composition furthercontains a pharmaceutically acceptable carrier. In certain embodimentsthat may be combined with any of the preceding embodiments, thetransplanted HSCs are human HSCs. In certain embodiments, the subject isin need or was in need of hematopoietic compartment autoreconstitution.In certain embodiments, the MYC-composition, Bcl-2-composition, or bothinduces endogenous hematopoietic stem cells (HSCs) to autoreconstitutethe hematopoietic compartment in the subject. In certain embodimentsthat may be combined with any of the preceding embodiments, thehematopoietic compartment autoreconstitution is enhanced compared tohematopoietic compartment autoreconstitution in a subject that is notadministered the composition containing a MYC-composition, aBcl-2-composition, or both. In certain embodiments that may be combinedwith any of the preceding embodiments, the enhanced hematopoieticcompartment autoreconstitution in the subject results in enhanced T cellcompartment autoreconstitution, enhanced B cell compartmentautoreconstitution, enhanced NK-cell compartment autoreconstitution,enhanced myeloid cell compartment autoreconstitution, or neutrophilrecovery. In certain embodiments that may be combined with any of thepreceding embodiments, administration of the composition containing aMYC-composition, a Bcl-2-composition, or both results in an expansion ofendogenous HSCs. In certain embodiments that may be combined with any ofthe preceding embodiments, the subject is undergoing or has undergonechemotherapy. In certain embodiments, administration of the compositioncontaining a MYC-composition, a Bcl-2-composition, or both prevents adecrease in hematopoietic compartment cells due to the chemotherapy. Incertain embodiments that may be combined with any of the precedingembodiments, administration of the composition containing aMYC-composition, a Bcl-2-composition, or both prevents a decrease in theamount of endogenous HSCs due to the chemotherapy. In certainembodiments that may be combined with any of the preceding embodiments,the subject is undergoing or has undergone radiation therapy. In certainembodiments, administration of the composition containing aMYC-composition, a Bcl-2-composition, or both prevents a decrease inhematopoietic compartment cells due to the radiation therapy. In certainembodiments that may be combined with any of the preceding embodiments,administration of the composition containing a MYC-composition, aBcl-2-composition, or both prevents a decrease in the amount ofendogenous HSCs due to the radiation therapy. In certain embodimentsthat may be combined with any of the preceding embodiments, the subjecthas a bone marrow failure syndrome. In certain embodiments, the bonemarrow failure syndrome is aplastic anemia or Gulf War Syndrome. Incertain embodiments, the bone marrow failure syndrome is an inheritedbone marrow failure syndrome (IBMFS). In certain embodiments, the IBMFSis selected from amegakaryocytic thrombocytopenia, Diamond-Blackfananemia, dyskeratosis congenita, fanconi anemia, Pearson syndrome, severecongenital neutropenia, Shwachman-Diamond syndrome, and thrombocytopeniaabsent radii, IVIC syndrome, WT syndrome, radio-ulnar synostosis, andataxia pancytopenia. In certain embodiments that may be combined withany of the preceding embodiments, administration of the compositioncontaining a MYC-composition, a Bcl-2-composition, or both prevents adecrease in hematopoietic compartment cells due to the bone marrowfailure syndrome. In certain embodiments that may be combined with anyof the preceding embodiments, administration of the compositioncontaining a MYC-composition, a Bcl-2-composition, or both prevents adecrease in the amount of endogenous HSCs due to the bone marrow failuresyndrome. In certain embodiments that may be combined with any of thepreceding embodiments, the MYC-composition is TAT-MYC. In certainembodiments that may be combined with any of the preceding embodiments,the therapeutically effective amount of the composition containing aMYC-composition is at least 0.1 mg/kg, at least 0.2 mg/kg, at least 0.3mg/kg, at least 0.4 mg/kg, at least 0.5 mg/kg, at least 0.6 mg/kg, atleast 0.7 mg/kg, at least 0.8 mg/kg, at least 0.9 mg/kg, at least 1mg/kg, at least 2 mg/kg, at least 3 mg/kg, at least 4 mg/kg, at least 5mg/kg, at least 6 mg/kg, at least 7 mg/kg, at least 8 mg/kg, at least 9mg/kg, at least 10 mg/kg, at least 20 mg/kg, at least 30 mg/kg, at least40 mg/kg, or at least 50 mg/kg of the subject's weight. In certainembodiments that may be combined with any of the preceding embodiments,the Bcl-2-composition is TAT-Bcl-2.

In certain embodiments that may be combined with any of the precedingembodiments, the therapeutically effective amount of the compositioncomprising a Bcl-2-composition is at least 0.1 mg/kg, at least 0.2mg/kg, at least 0.3 mg/kg, at least 0.4 mg/kg, at least 0.5 mg/kg, atleast 0.6 mg/kg, at least 0.7 mg/kg, at least 0.8 mg/kg, at least 0.9mg/kg, at least 1 mg/kg, at least 2 mg/kg, at least 3 mg/kg, at least 4mg/kg, at least 5 mg/kg, at least 6 mg/kg, at least 7 mg/kg, at least 8mg/kg, at least 9 mg/kg, at least 10 mg/kg, at least 20 mg/kg, at least30 mg/kg, at least 40 mg/kg, or at least 50 mg/kg of the subject'sweight. In certain embodiments that may be combined with any of thepreceding embodiments, the composition containing a MYC-composition, aBcl-2-composition, or both further contains a pharmaceuticallyacceptable carrier. In certain embodiments that may be combined with anyof the preceding embodiments, the composition containing aMYC-composition, the subject is a human patient. In certain embodimentsthat may be combined with any of the preceding embodiments, the subjectis a non-human animal.

Other aspects of the present disclosure relate to a use of a compositioncontaining a MYC-composition, a Bcl-2-composition, or both in a patientwho has had or will receive a hematopoietic stem cell (HSC) transplantto accelerate hematopoietic compartment reconstitution, where use of thecomposition achieves an at least 50% acceleration in hematopoieticcompartment reconstitution in the patient compared to hematopoieticcompartment reconstitution in a subject that does not receive thecomposition.

Other aspects of the present disclosure relate to a use of a compositioncontaining a MYC-composition, a Bcl-2-composition, or both in a patientwho has or has had a decrease in hematopoietic compartment cells toenhance hematopoietic compartment autoreconstitution, where theMYC-composition, Bcl-2-composition, or both induces endogenoushematopoietic stem cells (HSCs) to autoreconstitute the hematopoieticcompartment in the patient, and where use of the composition enhanceshematopoietic compartment autoreconstitution compared to hematopoieticcompartment autoreconstitution in a patient that does not receive thecomposition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict the results of FACS staining showing the developmentof T-cells in mice 4 weeks after transplant with expanded bone marrowcells and treatment with TAT-MYC. The panels show flow cytometry of TCRβpositive PBMCs. FIG. 1A and FIG. 1B depict flow cytometry of cells fromRag-1^(−/−) (FIG. 1A) and C57BL/6 (FIG. 1B) control mice that did notreceive a cell transplant or treatment with TAT-MYC. FIG. 1C and FIG. 1Ddepict flow cytometry of cells from Rag-1^(−/−) mice injected with 5×10³expanded bone marrow cells alone (FIG. 1C), or also receiving a 10 μginjection Tat-MYC 24 hours post-transplant (FIG. 1D).

FIGS. 2A-2D depict the results of FACS staining showing the developmentof B-cells in mice 4 weeks after transplant with expanded bone marrowcells and treatment with TAT-MYC. The panels show flow cytometry of B220positive PBMCs. FIG. 2A and FIG. 2B depict flow cytometry of cells fromRag-1^(−/−) (FIG. 2A) and C57BL/6 (FIG. 2B) control mice that did notreceive a cell transplant or treatment with TAT-MYC. FIG. 2C and FIG. 2Ddepict flow cytometry of cells from Rag-1^(−/−) mice injected with 5×10³expanded bone marrow cells alone (FIG. 2C), or also receiving a 10 μginjection Tat-MYC 24 hours post-transplant (FIG. 2D).

FIGS. 3A-3D depict the results of FACS staining showing the developmentof T-cells in mice 8 weeks after transplant with expanded bone marrowcells and treatment with TAT-MYC. The panels show flow cytometry of TCRβpositive PBMCs. FIG. 3A and FIG. 3B depict flow cytometry of cells fromRag-1^(−/−) (FIG. 3A) and C57BL/6 (FIG. 3B) control mice that did notreceive a cell transplant or treatment with TAT-MYC. FIG. 3C and FIG. 3Ddepict flow cytometry of cells from Rag-1^(−/−) mice injected with 5×10³expanded bone marrow cells alone (FIG. 3C), or also receiving a 10 μginjection Tat-MYC 24 hours post-transplant (FIG. 3D).

FIGS. 4A-4D depict the results of FACS staining showing the developmentof B-cells in mice 8 weeks after transplant with expanded bone marrowcells and treatment with TAT-MYC. The panels show flow cytometry of B220positive PBMCs. FIG. 4A and FIG. 4B depict flow cytometry of cells fromRag-1^(−/−) (FIG. 4A) and C57BL/6 (FIG. 4B) control mice that did notreceive a cell transplant or treatment with TAT-MYC. FIG. 4C and FIG. 4Ddepict flow cytometry of cells from Rag-1^(−/−) mice injected with 5×10³expanded bone marrow cells alone (FIG. 4C), or also receiving a 10 μginjection Tat-MYC 24 hours post-transplant (FIG. 4D).

FIGS. 5A-5D depict the results of FACS staining showing the developmentof T-cells in mice 4 weeks after transplant with freshly isolated wholebone marrow cells and treatment with TAT-MYC. The panels show flowcytometry of isolated PBMCs gated for CD8×CD4 positive cells. FIG. 5Aand FIG. 5B depict flow cytometry of cells from Rag-1^(−/−) (FIG. 5A)and C57BL/6 (FIG. 5B) control mice that did not receive a celltransplant or treatment with TAT-MYC. FIG. 5C and FIG. 5D depict flowcytometry of cells from Rag-1^(−/−) mice injected with 1×10⁶ whole bonemarrow cells alone (FIG. 5C), or also receiving a 10 μg injectionTat-MYC 24 hours post-transplant (FIG. 5D).

FIGS. 6A and 6B graphically depict the percentage of T cells for thefull cohort of mice shown in FIG. 5. FIG. 6A shows the % CD4 positive Tcells in C57BL/6 control mice (untreated; first column), Rag-1^(−/−)mice treated with 10⁶ BM cells only (middle column); and Rag-1^(−/−)mice treated with 10⁶ BM cells followed 24 hours later with 10 μgTAT-MYC (last column). FIG. 6B shows the % CD8 T cells in C57BL/6control mice (untreated; first column), Rag-1^(−/−) mice treated with10⁶ BM cells only (middle column); and Rag-1^(−/−) mice treated with 10⁶BM cells followed 24 hours later with 10 μg TAT-MYC (last column).

FIGS. 7A-7D depict the results of FACS staining showing the developmentof B-cells in mice 4 weeks after transplant with freshly isolated wholebone marrow cells and treatment with TAT-MYC. The panels show flowcytometry of isolated PBMCs gated for B220×CD19 positive cells. FIG. 7Aand FIG. 7B depict flow cytometry of cells from Rag-1^(−/−) (FIG. 7A)and C57BL/6 (FIG. 7B) control mice that did not receive a celltransplant or treatment with TAT-MYC. FIG. 7C and FIG. 7D depict flowcytometry of cells from Rag-1^(−/−) mice injected with 1×10⁶ whole bonemarrow cells alone (FIG. 7C), or also receiving a 10 μg injectionTat-MYC 24 hours post-transplant (FIG. 7D).

FIG. 8 graphically depicts the percentage of B cells for the full cohortof mice shown in FIG. 7. The graph shows the % CD19×B220 positive Bcells in C57BL/6 control mice (untreated; first column), Rag-1^(−/−)mice treated with 10⁶ BM cells only (middle column); and Rag-1^(−/−)mice treated with 10⁶ BM cells followed 24 hours later with 10 μgTAT-MYC (last column).

FIGS. 9A-9D graphically depict activation of splenic T-cells and B-cellsfrom Rag-1^(−/−) mice cohorts shown in FIGS. 5-8. FIG. 9A and FIG. 9Bshow the activation of T cells (FIG. 9A) and B cells (FIG. 9B) fromRag-1^(−/−) mice transplanted with 10⁶ fresh bone marrow cells, but nottreated with TAT-MYC. FIG. 9C and FIG. 9D show the activation of T cells(FIG. 9C) and B cells (FIG. 9D) from Rag-1^(−/−) mice treated with 10 μgTAT-MYC following transplantation with fresh bone marrow cells.

FIGS. 10A-10C depict the results of FACS staining showing thereconstitution of T-cells in mice 2 weeks after non-lethal challengewith 5FU followed by treatment with TAT-MYC. The panels show flowcytometry of isolated PBMCs gated for CD8×CD4 positive cells. FIG. 10Adepicts flow cytometry of cells from C57BL/6 control mice that did notreceive treatment with TAT-MYC. FIG. 10B depicts flow cytometry of cellsfrom C57BL/6 mice treated with 10 μg TAT-CRE. FIG. 10C depicts flowcytometry of cells from C57BL/6 mice treated with 10 μg Tat-MYC 24 hourspost-5FU challenge.

FIG. 11 graphically depicts the percentage of reconstituted CD4 T cellsfor the full cohort of 5FU challenged mice shown in FIG. 10. The graphshows the % CD4 positive T cells in C57BL/6 control mice (untreated;first column), C57BL/6 mice treated with 10 μg TAT-MYC (middle column);and C57BL/6 mice treated with 10 μg TAT-CRE (last column).

FIG. 12 graphically depicts the percentage of reconstituted CD8 T cellsfor the full cohort of 5FU challenged mice shown in FIG. 10. The graphshows the % CD8 positive T cells in C57BL/6 control mice (untreated;first column), C57BL/6 mice treated with 10 μg TAT-MYC (middle column);and C57BL/6 mice treated with 10 μg TAT-CRE (last column).

FIGS. 13A-13E depict the results of FACS staining showing thereconstitution of T-cells in mice 4 weeks after sub-lethal irradiationfollowed transplantation of freshly-isolated whole bone marrow andtreatment with TAT-MYC. The panels show flow cytometry of isolated PBMCsgated for CD4×TCRβ positive cells. FIG. 13A and FIG. 13B depict flowcytometry of cells from Rag-1^(−/−) (FIG. 13A) and C57BL/6 (FIG. 13B)control mice that did not receive a cell transplant or treatment withTAT-MYC. FIG. 13C, FIG. 13D, and FIG. 13E depict flow cytometry of cellsfrom Rag-1^(−/−) mice injected with 1×10⁶ whole bone marrow cells alone(FIG. 13C), or also receiving an intravenous (FIG. 13D) or intramuscular(FIG. 13E) injection of 10 μg Tat-MYC 24 hours post-transplant.

FIG. 14 graphically depicts the percentage of reconstituted CD4 T cellsfor the full cohort of 5FU challenged mice shown in FIG. 13. The graphshows the % CD4 positive T cells in Rag-1^(−/−) control mice (untreated;first column), Rag-1^(−/−) mice treated with 10 μg TAT-MYC intravenously(middle column); and Rag-1^(−/−) mice treated with 10 μg TAT-CREintramuscularly (last column).

FIGS. 15A-15E depict the results of FACS staining showing thereconstitution of B-cells in mice 4 weeks after sub-lethal irradiationfollowed by transplantation of freshly isolated whole bone marrow andtreatment with TAT-MYC. The panels show flow cytometry of isolated PBMCsgated for IgM×CD19 positive cells. FIG. 15A and FIG. 15B depict flowcytometry of cells from Rag-1^(−/−) (FIG. 15A) and C57BL/6 (FIG. 15B)control mice that did not receive a cell transplant or treatment withTAT-MYC. FIG. 15C, FIG. 15D, and FIG. 15E depict flow cytometry of cellsfrom Rag-1^(−/−) mice injected with 1×10⁶ whole bone marrow cells alone(FIG. 15C), or also receiving an intravenous (FIG. 15D) or intramuscular(FIG. 15E) injection of 10 μg Tat-MYC 24 hours post-transplant.

FIG. 16 graphically depicts the percentage of reconstituted B cells forthe full cohort of sublethally irradiated mice that were given HSCtransplants and then treated with either Tat-MYC or Tat-Cre, or acontrol 24 hours after the transplant shown in FIG. 15. The graph showsthe % CD19×B220 positive B cells in Rag-1^(−/−) control mice (untreated;first column), Rag-1^(−/−) mice treated with 10 μg TAT-MYC intravenously(middle column); and Rag-1^(−/−) mice treated with 10 μg TAT-CREintramuscularly (last column).

FIGS. 17A-17G depict shows a functional analysis of human cord bloodderived protein-transduced long term (ptlt)-HSC in vivo. FIG. 17Adepicts the results of FACS analysis showing reconstitution of the bonemarrow of cohorts of sublethally irradiated NSG mice given transplantsof 10⁶ cord blood cells expanded in vitro in a cocktail of cytokines(first panel; FCB), or expanded in a cocktail of cytokines supplementedwith Tat-Myc and Tat-Bcl-2 (second panel; FCB TMTB), or 5×10⁶ freshun-manipulated cord blood cells (third panel; Fresh FCB). FIG. 17Bdepicts the results of FACS analysis of bone marrow, spleen and thymuscells from the xenochimaeric mice reconstituted with ptlt-HSC shown inthe second panel of FIG. 17A. All cells were stained for human CD45.Gating on CD45+ cells showed human CD34+CD3810 cells in the bone marrow(first panel; BM); human CD19+ and human CD3+ lymphocytes in the spleen(second panel; spleen); and human CD3+ cells in the thymus (third panel;thymus). FIG. 17C depicts the results of FACS analysis of human splenicB cells that developed in the NSG mouse shown in the second panel ofFIG. 17A. The splenic B cells were labeled with CFSE and cultured in thepresence of monoclonal antibodies to human CD40 and IgM. Human B cellsfrom this mouse underwent proliferation following stimulation of theirantigen receptor. FIG. 17D shows a graphical representation of thequantification of myeloerythroid colonies (Burst Forming Unit Erythroid(BFU-E), Colony Forming Units Megakaryocyte (CFU-M), Colony FormingUnits Granulocyte (CFU-G), and Colony Forming Units Granulocyte Monocyte(CFU-GM)) from human CD34+ CD38^(lo) cells after plating onmethycellulose. The human CD34+ CD38^(lo) cells were obtained from thebone marrow of the NSG xenochimaeric mouse shown in FIG. 17A secondpanel further analyzed in FIG. 17B first panel. FIG. 17E shows agraphical representation of the quantification of the development ofmyeloerythroid colonies following replating on methycellulose. FIG. 17Fshows a graphical representation of the quantification of myeloid andlymphoid cell differentiation (CD11b, CD33, CD3, and CD19 expression) inthe CD45 positive population of bone marrow cells from the NSG mouseshown in FIG. 17A second panel. FIG. 17G shows a graphicalrepresentation of the quantification of myeloid and lymphoid celldifferentiation (CD11b, CD33, CD3, and CD19 expression) in the CD45positive population of spleen cells from the NSG mouse shown in FIG. 17Asecond panel.

FIGS. 18A-18F depict the results of FACS staining showing thereconstitution of the peripheral blood in NSG mice 8 weeks aftersub-lethal irradiation followed by transplantation of fresh fetal cordblood cells and treatment with TAT-MYC or Tat-Cre control protein. Thepanels show flow cytometry of isolated PBMCs gated for human CD45positive cells. FIGS. 18A-18C depict flow cytometry of cells from NSGmice that received 5×10⁵ (FIG. 18A), 1×10⁶ (FIG. 18B), or 5×10⁶ (FIG.18C) freshly isolated fetal cord blood cells followed 24 hours later byan injection of 10 Tat-Cre. FIGS. 18D-18F depict flow cytometry of cellsfrom NSG mice that received 5×10⁵ (FIG. 18D), 1×10⁶ (FIG. 18E), or 5×10⁶(FIG. 18F) freshly isolated fetal cord blood cells followed 24 hourslater by an injection of 10 μg Tat-MYC.

FIGS. 19A-19F depict the results of FACS staining showing thereconstitution of the bone marrow from NSG mice 8 weeks after sub-lethalirradiation followed transplantation of fresh fetal cord blood cells andtreatment with TAT-MYC or Tat-Cre control protein. The panels show flowcytometry of isolated bone marrow gated for human CD45 positive cells.FIGS. 19A-19C depict flow cytometry of cells from NSG mice that received5×10⁵ (FIG. 19A), 1×10⁶ (FIG. 19B), or 5×10⁶ (FIG. 19C) freshly isolatedfetal cord blood cells followed 24 hours later by an injection of 10 μgTat-Cre. FIGS. 19D-19F depict flow cytometry of cells from NSG mice thatreceived 5×10⁵ (FIG. 19D), 1×10⁶ (FIG. 19E), or 5×10⁶ (FIG. 19F) freshlyisolated fetal cord blood cells followed 24 hours later by an injectionof 10 μg Tat-MYC.

FIGS. 20A-20F depict the results of FACS staining showing thereconstitution of the spleen in NSG mice 8 weeks after sub-lethalirradiation followed transplantation of fresh fetal cord blood cells andtreatment with TAT-MYC or Tat-Cre control protein. The panels show flowcytometry of isolated spleen cells gated for human CD45 positive cells.FIGS. 20A-20C depict flow cytometry of cells from NSG mice that received5×10⁵ (FIG. 20A), 1×10⁶ (FIG. 20B), or 5×10⁶ (FIG. 20C) freshly isolatedfetal cord blood cells followed 24 hours later by an injection of 10 μgTat-Cre. FIGS. 20D-20F depict flow cytometry of cells from NSG mice thatreceived 5×10⁵ (FIG. 20D), 1×10⁶ (FIG. 20E), or 5×10⁶ (FIG. 20F) freshlyisolated fetal cord blood cells followed 24 hours later by an injectionof 10 μg Tat-MYC.

FIGS. 21A and 21B depict the results of FACS staining showing thereconstitution of the peripheral blood in NSG mice 8 weeks aftersub-lethal irradiation followed transplantation of fresh fetal cordblood cells that were treated in vitro for 1 hour with TAT-MYC prior toinjecting into mice. The panels show flow cytometry of isolated PBMCsgated for human CD45 positive cells. FIG. 21A depicts flow cytometry ofcells from NSG mice that received 5×10⁶ freshly isolated fetal cordblood cells. FIG. 21B depicts flow cytometry of cells from NSG mice thatreceived 5×10⁶ freshly isolated fetal cord blood cells treated in vitrowith Tat-MYC for 1 hour prior to injecting the cells into mice.

FIGS. 22A-22F depict the results of FACS staining showing the long-termreconstitution of the bone marrow, spleen and thymus in NSG mice 8months after sub-lethal irradiation followed transplantation of freshfetal cord blood cells that were treated in vitro for 1 hour withTAT-MYC prior to injecting into mice. The panels show flow cytometry ofisolated bone marrow, spleen and thymus cells gated for human CD45positive cells. FIG. 22A depicts flow cytometry of bone marrow cellsfrom NSG mice that received 5×10⁶ freshly isolated fetal cord bloodcells. FIG. 22B depicts flow cytometry of bone marrow cells from NSGmice that received 5×10⁶ freshly isolated fetal cord blood cells treatedin vitro with Tat-MYC for 1 hour prior to injecting the cells into mice.FIG. 22C depicts flow cytometry of spleen cells from NSG mice thatreceived 5×10⁶ freshly isolated fetal cord blood cells. FIG. 22D depictsflow cytometry of Spleen cells from NSG mice that received 5×10⁶ freshlyisolated fetal cord blood cells treated in vitro with Tat-MYC for 1 hourprior to injecting the cells into mice. FIG. 22E depicts flow cytometryof thymus cells from NSG mice that received 5×10⁶ freshly isolated fetalcord blood cells. FIG. 22F depicts flow cytometry of thymus cells fromNSG mice that received 5×10⁶ freshly isolated fetal cord blood cellstreated in vitro with Tat-MYC for 1 hour prior to injecting the cellsinto mice.

FIGS. 23A-23C depict a FACS analysis of the peripheral blood from acontrol NSG mouse (FIG. 23A), a sublethally irradiated NSG mouse giventransplants of 5×10⁶ C-GSF mobilized adult blood cells expanded in vitroin a cocktail of cytokines (FIG. 23B), or 5×10⁶ C-GSF mobilized adultblood cells expanded in a cocktail of cytokines supplemented withTat-MYC and Tat-Bcl-2 (FIG. 23C).

FIG. 24 depicts the amino acid and nucleic acid sequences for someembodiments of the Tat-Myc polypeptide.

FIG. 25 depicts the amino acid and nucleic acid sequences for someembodiments of the Bcl-2 domain polypeptide.

DETAILED DESCRIPTION

The present disclosure relates, in part, to enhancing hematopoieticcompartment cell formation in a subject in need thereof by administeringa composition containing a MYC-composition, such as a proteintransduction domain-MYC (PTD-MYC) fusion protein, a compositioncontaining a Bcl-2-composition, such as a protein transductiondomain-Bcl-2 (PTD-Bcl-2) fusion protein, or both. In certain aspects,the present disclosure relates to accelerating hematopoietic stem cell(HSC) engraftment and hematopoietic compartment reconstitution in an HSCtransplant recipient by administering a composition containing aMYC-composition, such as a protein transduction domain-MYC (PTD-MYC)fusion protein, a composition containing a Bcl-2-composition, such as aprotein transduction domain-Bcl-2 (PTD-Bcl-2) fusion protein, or both.In some embodiments, the HSC transplant recipient is also administered acomposition containing a MYC-composition, such as a protein transductiondomain-MYC (PTD-MYC) fusion protein, a composition containing aBcl-2-composition, such as a protein transduction domain-Bcl-2(PTD-Bcl-2) fusion protein, or both to maintain and/or furtheraccelerate HSC engraftment and hematopoietic compartment reconstitutionin the HSC transplant recipient. As used herein, HSC transplantationincludes, without limitation, bone marrow transplantation.

Moreover, the present disclosure is based, at least in part, on thediscovery that administering a composition having a fusion proteincontaining a MYC polypeptide and a PTD, such as the HIV TAT proteintransduction domain, after bone transplantation reduced the timerequired for recovery of mature hematopoietic lineages after bone marrowtransplantation. For example, it was shown that the time required forT-cell recovery was reduced from about 9 weeks to about 4 weeks, and thetime required for B-cell recovery was reduced from about 10 weeks toabout 4 weeks in lethally irradiated mice that were administered aMYC-composition after bone marrow transplantation (Example 1).Accordingly, one aspect of the present disclosure provides methods ofaccelerating hematopoietic compartment reconstitution afterhematopoietic stem cell (HSC) transplantation in a subject, by: a)administering a therapeutically effective amount of a first compositioncontaining HSCs to achieve hematopoietic compartment reconstitution in asubject in need thereof; and b) administering a second compositioncontaining a MYC-composition, a Bcl-2-composition, or both to thesubject, where administering the second composition achieves an at least50% acceleration in hematopoietic compartment reconstitution compared tohematopoietic compartment reconstitution in a subject that is notadministered the second composition.

In other aspects, the present disclosure relates to enhancinghematopoietic compartment autoreconstitution in a subject in needthereof by administering a composition having a protein transductiondomain-MYC (PTD-MYC) fusion protein. The present disclosure is alsobased, at least in part, on the novel discovery that administration of aMYC-composition can enhance recovery in a subject undergoingchemotherapy and/or radiation therapy after suffering a decreasehematopoietic compartment cells due to the therapy. For example, it wasshown that administration of a MYC-composition to a subject aftertreatment with the chemotherapeutic drug 5-fluorouracil acceleratedrecovery of hematopoietic compartment cells (e.g., CD8⁻ T-cells)following the reduction due to the 5-fluorouracil treatment (FIG. 14).

Another aspect of the present disclosure provides methods of enhancinghematopoietic compartment autoreconstitution in a subject, by:administering a therapeutically effective amount of a compositioncontaining a MYC-composition, a composition containing aBcl-2-composition, or both to a subject in need of hematopoieticcompartment autoreconstitution, where the MYC-composition, theBcl-2-composition, or both induces endogenous HSCs to autoreconstitutethe hematopoietic compartment in the subject, and where hematopoieticcompartment autoreconstitution is enhanced compared to hematopoieticcompartment autoreconstitution in a subject that is not administered thecomposition.

In other aspects, the present disclosure relates, in part, to enhancinghematopoietic compartment reconstitution in a subject in need ofhematopoietic stem cell transplantation by pre-treating a population ofHSCs with a MYC-composition, such as a protein transduction domain-MYC(PTD-MYC) fusion protein; a Bcl-2 composition, such as a PTD-Bcl-2fusion protein; or both, prior to administering the HSCs to the subject.In certain aspects, the present disclosure relates to acceleratinghematopoietic stem cell (HSC) engraftment and hematopoietic compartmentreconstitution in an HSC transplant recipient by pre-treating HSCs witha protein transduction domain-MYC (PTD-MYC) fusion protein, a proteintransduction domain-Bcl-2 (PTD-Bcl-2) fusion protein, or both prior toadministering the HSCs to the subject. Surprisingly, pre-treating HSCsfor at least as little as 1 hour prior to transplantation (and perhapsas little as about 10 minutes) is sufficient for the MYC-composition,the Bcl-2 composition, or both to achieve an enhancement inhematopoietic compartment reconstitution.

Accordingly, certain preferred embodiments relate to methods ofenhancing hematopoietic compartment reconstitution to a subject in needof hematopoietic stem cell transplantation, by treating a population ofhematopoietic stem cells with a composition containing aMYC-composition, a composition containing a Bcl-2-composition, or both,for less than about 13 days; and administering to the subject, atherapeutically effective amount of the treated population ofhematopoietic stem cells to reconstitute the hematopoietic compartmentof the subject, wherein hematopoietic compartment reconstitution isenhanced compared to hematopoietic compartment reconstitution in asubject that is administered a population of hematopoietic stem cellsthat were not treated with the composition containing a MYC-composition,the composition containing a Bcl-2-composition, or both. In someembodiments, the population of hematopoietic stem cells is treated withthe composition containing a MYC-composition, the composition containinga Bcl-2-composition, or both, for less than about or about 12 days toless than about or about 1 days, for example, less than about or about12 days, less than about or about 11 days, less than about or about 10days, less than about or about 9 days, less than about or about 8 days,less than about or about 7 days, less than about or about 6 days, lessthan about or about 5 days, less than about or about 4 days, less thanabout or about 2 days, or less than about or about 1 day. In someembodiments, the population of hematopoietic stem cells is treated withthe composition containing a MYC-composition, the composition containinga Bcl-2-composition, or both, for less than about or about 24 hours toless than about or about 1 hour, for example, less than about or about24 hours, less than about or about 23 hours, less than about or about 22hours, less than about or about 21 hours, less than about or about 20hours, less than about or about 19 hours, less than about or about 18hours, less than about or about 17 hours, less than about or about 16hours, less than about or about 15 hours, less than about or about 14hours, less than about or about 13 hours, less than about or about 12hours, less than about or about 11 hours, less than about or about 10hours, less than about or about 9 hours, less than about or about 8hours, less than about or about 7 hours, less than about or about 6hours, less than about or about 5 hours, less than about or about 4hours, less than about or about 3 hours, less than about or about 2hours, or less than about or about 1 hour. In some embodiments, thepopulation of hematopoietic stem cells is treated with the compositioncontaining a MYC-composition, the composition containing aBcl-2-composition, or both, for less than about or about 60 minutes toless than about or about 10 minutes, for example, less than about orabout 60 minutes, less than about or about 55 minutes, less than aboutor about 50 minutes, less than about or about 45 minutes, less thanabout or about 40 minutes, less than about or about 35 minutes, lessthan about or about 30 minutes, less than about or about 29 minutes,less than about or about 28 minutes, less than about or about 27minutes, less than about or about 26 minutes, less than about or about25 minutes, less than about or about 24 minutes, less than about orabout 23 minutes, less than about or about 22 minutes, less than aboutor about 21 minutes, less than about or about 20 minutes, less thanabout or about 19 minutes, less than about or about 18 minutes, lessthan about or about 17 minutes, less than about or about 16 minutes,less than about or about 15 minutes, less than about or about 14minutes, less than about or about 13 minutes, less than about or about12 minutes, less than about or about 11 minutes, or less than about orabout 10 minutes.

As used herein, the term “hematopoietic compartment” refers to the cellcompartment in a subject that contains all blood cell lineages,including without limitation, the myeloid lineage, which includes,without limitation, monocytes, macrophages, neutrophils, basophils,eosinophils, erythrocytes, megakaryocytes, platelets, and dendriticcells; and the lymphoid lineage, which includes, without limitation,T-cells, B-cells, NKT-cells, and NK cells. The “hematopoieticcompartment” can contain all immature, mature, undifferentiated, anddifferentiated white blood cell populations and sub-populations,including tissue-specific and specialized varieties.

As used herein, the term “hematopoietic compartment cell formation” in asubject refers to the production and/or expansion of one or more cellsof any blood cell lineages of the hematopoietic compartment in thehematopoietic compartment from hematopoietic stem cell (HSC)differentiation, HSC proliferation, and/or HSC survival. “Hematopoieticcompartment cell formation” may be the result of HSC engraftment byexogenous HSCs, such as hematopoietic compartment reconstitution in anHSC transplant recipient. Alternatively, hematopoietic compartment cellformation” may be the result of endogenous HSC differentiation,endogenous HSC proliferation, and/or endogenous HSC survival, such asfrom hematopoietic compartment autoreconstitution in a subject. In someembodiments, “hematopoietic compartment cell formation” includes,without limitation, one or more of myeloid lineage formation, myeloidlineage progenitor cell formation, monocyte cell formation, macrophagecell formations, neutrophil cell formation, basophil cell formation,eosinophil cell formation, erythrocyte cell formation, megakaryocytecell formation, platelet cell formation, dendritic cell formation,lymphoid lineage formation, lymphoid lineage progenitor cell formation,T-cell formation, B-cell formation, NKT-cell formation, and NK cellformation.

As used herein, “enhancing hematopoietic compartment cell formation” ina subject refers to one or more of: i) increasing the rate ofhematopoietic compartment cell formation (e.g., acceleratinghematopoietic compartment reconstitution with exogenous HSCs oraccelerating autoreconstitution with endogenous HSCs) by at least aboutor about 5% to at least about or about 500%, as compared to the rate ofhematopoietic compartment cell formation in a subject that isadministered exogenous HSCs that have not been treated with aMYC-composition, a Bcl-2 composition, or both, or as compared to therate of hematopoietic compartment cell formation from HSCs in a subjectthat is not administered a MYC-composition; ii) increasing the amount ofhematopoietic compartment cells that are formed in the hematopoieticcompartment of the subject from either exogenous or endogenous HSCs byat least about or about 5% to at least about or about 500%, as comparedto the amount of hematopoietic compartment cells that are formed in thehematopoietic compartment in a subject that is administered exogenousHSCs that have not been treated with a MYC-composition, a Bcl-2composition, or both, or as compared to the amount of hematopoieticcompartment cells that are formed in the hematopoietic compartment in asubject that is not administered a MYC-composition, a Bcl-2 composition,or both; or iii) reducing loss of hematopoietic compartment cells in thesubject by at least about or about 5% to at least about or about 500%,as compared to the amount of hematopoietic compartment cells that arelost in a subject that is administered exogenous HSCs that have not beentreated with a MYC-composition, a Bcl-2 composition, or both, or ascompared to the amount of hematopoietic compartment cells that are lostin a subject that is not administered a MYC-composition, a Bcl-2composition or both. Moreover, “enhancing hematopoietic compartment cellformation” in a subject includes, without limitation, enhancinghematopoietic compartment reconstitution from exogenous HSCs (e.g., HSCengraftment), and enhancing hematopoietic compartment autoreconstitutionfrom endogenous HSCs. As used herein, “loss of hematopoietic compartmentcells” in a subject refers to a reduction in the amount of hematopoieticcompartment cells due to cell necrosis, apoptosis, and the like.

Similarly, hematopoietic compartment cell formation in a subject isenhanced when: i) the rate of hematopoietic compartment cell formationis increased (e.g., hematopoietic compartment reconstitution withexogenous HSCs is accelerated or autoreconstitution with endogenous HSCsis accelerated), for example, by at least about or about 5% to at leastabout or about 500%, as compared to the rate of hematopoieticcompartment cell formation in a subject that is administered exogenousHSCs that have not been treated with a MYC-composition, aBcl-2-composition, or both, or as compared to the rate of hematopoieticcompartment cell formation from HSCs in a subject that is notadministered a MYC-composition; ii) the amount of hematopoieticcompartment cells that are formed in the hematopoietic compartment ofthe subject is increased, for example, by at least about or about 5% toat least about or about 500%, as compared to the amount of hematopoieticcompartment cells that are formed in the hematopoietic compartment in asubject that is administered exogenous HSCs that have not been treatedwith a MYC-composition, a Bcl-2-composition, or both, or as compared tothe amount of hematopoietic compartment cells that are formed in thehematopoietic compartment in a subject that is not administered aMYC-composition; and/or iii) loss of hematopoietic compartment cells inthe subject is reduced, for example, by at least about or about 5% to atleast about or about 500%, as compared to the amount of hematopoieticcompartment cells that are lost in a subject that is administeredexogenous HSCs that have not been treated with a MYC-composition, aBcl-2-composition, or both, or as compared to the amount ofhematopoietic compartment cells that are lost in a subject that is notadministered a MYC-composition.

Any method known in the art and disclosed herein for measuring the rateof hematopoietic compartment cell formation (e.g., hematopoieticcompartment reconstitution or autoreconstitution) from HSCs, formeasuring the amount of hematopoietic compartment cells that are formedin the hematopoietic compartment of a subject, and/or for measuring lossof hematopoietic compartment cells in a subject may be used. In onenon-limiting example, fluorescent-tagged antibodies specific for bloodcell lineage marker and fluorescence-activated flow cytometry (FACS)analysis is utilized.

In certain embodiments, the rate of hematopoietic compartment cellformation is considered to be increased when the rate of hematopoieticcompartment cell formation is increased, for example, by at least aboutor about 5%, at least about or about 10%, at least about or about 15%,at least about or about 20%, at least about or about 25%, at least aboutor about 30%, at least about or about 31%, at least about or about 32%,at least about or about 33%, at least about or about 34%, at least aboutor about 35%, at least about or about 40%, at least about or about 45%,at least about or about 50%, at least about or about 55%, at least aboutor about 60%, at least about or about 65%, at least about or about 66%,at least about or about 67%, at least about or about 68%, at least aboutor about 69%, at least about or about 70%, at least about or about 75%,at least about or about 80%, at least about or about 90%, at least aboutor about 95%, at least about or about 100%, at least about or about150%, at least about or about 200%, at least about or about 250%, atleast about or about 300%, at least about or about 400%, at least aboutor about 500%, or a higher percentage, as compared to the rate ofhematopoietic compartment cell formation in a subject that isadministered exogenous HSCs that have not been treated with aMYC-composition, a Bcl-2-composition, or both, or as compared to therate of hematopoietic compartment cell formation from HSCs in a subjectthat is not administered a MYC-composition.

In certain embodiments, the amount of hematopoietic compartment cellsthat are formed in the hematopoietic compartment of a subject isconsidered to be increased when the amount of hematopoietic compartmentcells formed is increased, for example, by at least about or about 5%,at least about or about 10%, at least about or about 15%, at least aboutor about 20%, at least about or about 25%, at least about or about 30%,at least about or about 31%, at least about or about 32%, at least aboutor about 33%, at least about or about 34%, at least about or about 35%,at least about or about 40%, at least about or about 45%, at least aboutor about 50%, at least about or about 55%, at least about or about 60%,at least about or about 65%, at least about or about 66%, at least aboutor about 67%, at least about or about 68%, at least about or about 69%,at least about or about 70%, at least about or about 75%, at least aboutor about 80%, at least about or about 90%, at least about or about 95%,at least about or about 100%, at least about or about 150%, at leastabout or about 200%, at least about or about 250%, at least about orabout 300%, at least about or about 400%, at least about or about 500%,or a higher percentage, as compared to the amount of hematopoieticcompartment cells that are formed in the hematopoietic compartment in asubject that is administered exogenous HSCs that have not been treatedwith a MYC-composition, a Bcl-2-composition, or both, or as compared tothe amount of hematopoietic compartment cells that are formed in thehematopoietic compartment in a subject that is not administered aMYC-composition.

In certain embodiments, loss of hematopoietic compartment cells in asubject is considered to be decreased when the loss of cells is reduced,for example, by at least about or about 5%, at least about or about 10%,at least about or about 15%, at least about or about 20%, at least aboutor about 25%, at least about or about 30%, at least about or about 31%,at least about or about 32%, at least about or about 33%, at least aboutor about 34%, at least about or about 35%, at least about or about 40%,at least about or about 45%, at least about or about 50%, at least aboutor about 55%, at least about or about 60%, at least about or about 65%,at least about or about 66%, at least about or about 67%, at least aboutor about 68%, at least about or about 69%, at least about or about 70%,at least about or about 75%, at least about or about 80%, at least aboutor about 90%, at least about or about 95%, at least about or about 100%,at least about or about 150%, at least about or about 200%, at leastabout or about 250%, at least about or about 300%, at least about orabout 400%, at least about or about 500%, or a higher percentage, ascompared to the amount of hematopoietic compartment cells that are lostin a subject that is administered exogenous HSCs that have not beentreated with a MYC-composition, a Bcl-2-composition, or both, or ascompared to the amount of hematopoietic compartment cells that are lostin a subject that is not administered a MYC-composition.

MYC-Compositions

Certain aspects of the present disclosure relate to treating apopulation of hematopoietic stem cells (HSCs) with a compositioncontaining a MYC-composition to enhance hematopoietic compartmentreconstitution. HSCs of the present disclosure may be treated with theMYC-composition alone, or in combination with a Bcl-2-composition of thepresent disclosure. Any method of treating cells with a composition,such as fusion protein, known in the art and disclosed herein may beused. For example, a population of hematopoietic stem cells may becultured in the presence of the MYC-composition. Other aspect of thepresent disclosure relate to administering to a subject in need thereof,a composition containing a MYC-composition to enhance hematopoieticcompartment formation in the subject.

As used herein, a “MYC-composition” refers to a MYC polypeptide; avariant or mutant of a MYC polypeptide, a modified MYC polypeptide, ahomologue of a MYC polypeptide; an analogue of a MYC polypeptide; abiologically active fragment of a MYC polypeptide; a downstream targetof a MYC polypeptide, a homologue thereof, an analogue thereof, or abiologically active fragment thereof; and a fusion protein containing aMYC polypeptide, a homologue thereof, an analogue thereof, and abiologically active fragment thereof. A MYC-composition of the presentdisclosure includes any MYC polypeptide, variant thereof, mutantthereof, homologue thereof, analogue thereof, or biologically activefragment thereof known in the art (e.g., US Patent ApplicationPublication Nos. US 2007/0116691, US 2009/0291094, US 2010/0047217, US2010/0055129, and US 2010/0279351).

In certain preferred embodiments, MYC-compositions of the presentdisclosure are fusion proteins that contain a MYC polypeptide, variantthereof, mutant thereof, homologue thereof, analogue thereof, orbiologically active fragment thereof that has been coupled (e.g., fused)to a protein transduction domain (PTD).

MYC Polypeptides

In some embodiments, a MYC-composition of the present disclosure is aMYC polypeptide. A MYC polypeptide of the present disclosure includes,without limitation, any polypeptide having one or more activities of afull-length MYC protein.

As used herein, “MYC” and “MYC protein” are used interchangeably andrefer to a protein that is a member of the MYC family of bHLH (basichelix-loop-helix) transcription factors. MYC proteins of the presentdisclosure are transcription factors that regulate expression of MYCresponsive genes, and as such are required to enter the nucleus of acell to function as transcription factors. MYC activity can activateexpression of certain MYC responsive genes, while repressing expressionof other MYC responsive genes. MYC activity can regulate variouscellular functions including, without limitation, cell proliferation,cell growth, and apoptosis.

MYC-compositions of the present disclosure allow for an increase in MYCactivity in a subject in need of hematopoietic compartment cellformation by the exogenous addition of MYC, without the need foroverexpressing endogenous MYC or recombinantly expressing MYC viagenetic manipulation.

MYC polypeptides of the present disclosure include, without limitation,full-length MYC proteins, fragments of MYC proteins that retain at leastone activity of a full-length MYC protein, homologs thereof that retainat least one activity of a full-length MYC protein, and analogs thereofthat retain at least one activity of a full-length MYC protein. MYCpolypeptides of the present disclosure may be produced by any suitablemethod known in the art. For example, a MYC polypeptide may be purifiedfrom a native source, may be recombinantly expressed, or may bechemically synthesized.

MYC Proteins

Examples of full-length MYC proteins suitable for use in any of themethods of the present disclosure include, without limitation, c-Myc,N-Myc, L-Myc, and S-Myc.

In certain preferred embodiments, the MYC polypeptide is a full-lengthc-Myc polypeptide. The c-Myc polypeptide may have one or more of thefollowing features: the polypeptide may be a polymer of 439 amino acids,the polypeptide may have a molecular weight of 48,804 kDa, thepolypeptide may contain a basic Helix-Loop-Helix Leucine Zip-per(bHLH/LZ) domain, or the polypeptide may bind to a sequence containingCACGTG (i.e., an E-box sequence). Preferably, the c-Myc polypeptide isthe human c-Myc polypeptide having NCBI Accession Number NP_002458.2.Moreover, a c-Myc polypeptide of the present disclosure may be a c-Mycpolypeptide that has not undergone any post-translational modifications.Alternatively, a c-Myc polypeptide of the present disclosure may be ac-Myc polypeptide that has undergone post-translational modifications.

In some embodiments, the MYC polypeptide is a fusion protein containinga protein transduction domain (PTD). In certain embodiments, the MYCpolypeptide is a fusion protein containing a TAT protein, or fragmentthereof, of the present disclosure. In some embodiments, the MYCpolypeptide is a TAT-MYC fusion protein with the following amino acidsequence (SEQ ID NO: 1):

MRKKRRQRRRMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRKGELNSKLEGKPIPNPLLGLDSTRTGHHHHHH

In the TAT-MYC polypeptide of SEQ ID NO: 1, amino acids 2-10 correspondto the HIV TAT protein transduction domain, amino acids 11-454correspond to the amino acid sequence of c-Myc, amino acids 455-468correspond to a 14 amino acid V5 epitope, and amino acids 472-477correspond to a 6 Histidine tag.

Biologically Active MYC Fragments

In other embodiments, a MYC-composition of the present disclosure is abiologically active fragment of a full-length MYC protein that retainsat least one activity of a full-length MYC protein. The MYC polypeptidemay be a fragment of c-Myc, N-Myc, L-Myc, or S-Myc.

A MYC fragment of the present disclosure may contain at least 10, atleast 15, at least 20, at least 25, at least 30, at least 35, at least40, at least 45, at least 50, at least 55, at least 60, at least 65, atleast 70, at least 75, at least 80, at least 85, at least 90, at least95, at least 100, at least 150, at least 200, at least 250, at least300, at least 350, at least 400, or more consecutive amino acid residuesof the amino acid sequence of a MYC protein.

MYC Homologues

In other embodiments, a MYC-composition of the present disclosure is ahomologue of a MYC protein, or a homologue of a fragment thereof thatretains at least one activity of a full-length MYC protein.

For example, a MYC polypeptide of the present disclosure may include anamino acid sequence that is at least 40% to 100% identical, e.g., atleast 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percent from about40% to about 100% identical to a MYC protein or fragments thereof. Incertain embodiments, the MYC polypeptide is a homologue of c-Myc, N-Myc,L-Myc, S-Myc, or fragments thereof.

MYC polypeptides of the present disclosure also include functionalhomologs or analogs of the human c-Myc polypeptide having NCBI AccessionNumber NP_002458.2, or fragment thereof. In certain embodiments, thec-Myc homolog or analog contains an amino acid sequence that is at least40% to 100% identical, e.g., at least 40%, 45%. 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, orany other percent from about 40% to about 100% identical to the c-Mycpolypeptide sequence of NCBI Accession Number NP_002458.2 or a fragmentthereof.

In other embodiments, the c-Myc homolog or analog contains a polypeptidesequence of at least 10 amino acids, at least 20 amino acids, at least30 amino acids, at least 40 amino acids, at least 50 amino acids, atleast 60 amino acids, at least 70 amino acids, at least 80 amino acids,at least 90 amino acids, at least 100 amino acids, at least 150 aminoacids, at least 200 amino acids, at least 250 amino acids, at least 300amino acids, at least 350 amino acids, at least 400 amino acids, or morein length that is at least 50% to 100% identical, e.g., at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%,95%, 96%, 97%, 98%, or any other percent from about 50% to about 100%identical to the c-Myc polypeptide sequence of NCBI Accession NumberNP_002458.2 or a fragment thereof.

As used herein, a “homologue” refers to a protein or polypeptide havingamino acid sequence similarity between a reference sequence and at leasta fragment of a second sequence. Homologues may be identified by anymethod known in the art, preferably, by using the BLAST tool to comparea reference sequence to a single second sequence or fragment of asequence or to a database of sequences. As described below, BLAST willcompare sequences based upon percent identity and similarity.

The terms “identical” or percent “identity,” in the context of two ormore sequences (e.g., amino acid sequences), refer to two or moresequences or subsequences that are the same. Two sequences aresubstantially identical if two sequences have a specified percentage ofamino acid residues or nucleotides that are the same (i.e., 29%identity, optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 99% or 100% identity over a specified region, or, whennot specified, over the entire sequence), when compared and aligned formaximum correspondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Optionally, the identity existsover a region that is at least about 10 amino acids in length, or morepreferably over a region that is 20, 50, 200, or more amino acids inlength.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. When comparing two sequences foridentity, it is not necessary that the sequences be contiguous, but anygap would carry with it a penalty that would reduce the overall percentidentity. For blastp, the default parameters are Gap opening penalty=11and Gap extension penalty=1. For blastn, the default parameters are Gapopening penalty=5 and Gap extension penalty=2.

As used herein, a “comparison window” includes reference to a segment ofany one of the number of contiguous positions including, but not limitedto from 20 to 600, usually about 50 to about 200, more usually about 100to about 150 in which a sequence may be compared to a reference sequenceof the same number of contiguous positions after the two sequences areoptimally aligned. Methods of alignment of sequences for comparison arewell known in the art. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith andWaterman (1981), by the homology alignment algorithm of Needleman andWunsch (1970) J Mol Biol 48(3):443-453, by the search for similaritymethod of Pearson and Lipman (1988) Proc Natl Acad Sci USA85(8):2444-2448, by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or bymanual alignment and visual inspection [see, e.g., Brent et al., (2003)Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (RingbouEd)].

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nucleic AcidsRes 25(17):3389-3402 and Altschul et al. (1990) J. Mol Biol215(3)-403-410, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always>0)and N (penalty score for mismatching residues; always<0). For amino acidsequences, a scoring matrix is used to calculate the cumulative score.Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. For aminoacid sequences, the BLASTP program uses as defaults a wordlength of 3,and expectation (E) of 10, and the BLOSUM62 scoring matrix [see Henikoffand Henikoff, (1992) Proc Natl Acad Sci USA 89(22):10915-10919]alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands. For nucleotide sequences, the BLASTN program uses asdefaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4,and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, (1993)Proc Natl Acad Sci USA 90(12):5873-5877).

One measure of similarity provided by the BLAST algorithm is thesmallest sum probability (P(N)), which provides an indication of theprobability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a nucleic acid isconsidered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.2, more preferably less than about0.01, and most preferably less than about 0.001.

Other than percentage of sequence identity noted above, anotherindication that two polypeptides are substantially identical is that thefirst polypeptide is immunologically cross-reactive with antibodiesraised against the second polypeptide. Thus, a polypeptide is typicallysubstantially identical to a second polypeptide, for example, where thetwo peptides differ only by conservative substitutions.

As disclosed herein, suitable MYC polypeptides also includeconservatively modified variants of MYC polypeptides of the presentdisclosure. “Conservatively modified variants” as used herein includeindividual substitutions, deletions, or additions to an encoded aminoacid sequence which result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.The following eight groups contain amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Glycine (G); 2) Asparticacid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4)Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine(M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7)Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see,e.g., Creighton, Proteins (1984)).

MYC Analogues,

In other embodiments, a MYC-composition of the present disclosure is ananalogue of a MYC protein, or an analogue of a fragment thereof thatretains at least one activity of a full-length MYC protein. In certainembodiments, the MYC polypeptide is an analogue of c-Myc, N-Myc, L-Myc,S-Myc, or fragments thereof.

As used herein, an “analogue” refers to a protein that structurally orfunctionally resembles a protein of interest, such as a MYC polypeptide.Compared to the starting protein, an analogue may exhibit the same,similar, or improved activity and/or function. Methods of screening foranalogues and/or synthesizing analogues are well known in the art.

Suitable MYC analogues of the present disclosure include, withoutlimitation, HIF-1a and ICN-1.

Proteins Downstream of MYC

In other embodiments, a MYC-composition of the present disclosure is aprotein that is downstream of MYC in a MYC pathway. Any proteindownstream known in the art is suitable for use with the methods of thepresent disclosure. Examples of suitable proteins that are downstream ofMYC include, without limitation, AKT and AKT-related proteins, such asPDK-1, mTORC2, PI3K-delta. The protein downstream of MYC t may furtherinclude a protein transduction domain (PTD). Accordingly, in certainembodiments, the protein downstream of MYC is an AKT-PTD fusion protein,a PTD-PDK-1 fusion protein, a PTD-mTORC2 fusion protein, or aPTD-PI3K-delta fusion protein.

In other embodiments, hematopoietic compartment reconstitution isenhanced in a subject in need thereof by administering HSCs that havebeen pre-treated with an inhibitor (e.g., genetic, chemical, smallmolecule, etc.) that inhibits a protein that antagonizes HSC survivaland/or proliferation and that is downstream of MYC. Similarly,hematopoietic compartment formation is enhanced in a subject in needthereof by administering a therapeutically effective amount of acomposition containing inhibitor (e.g., genetic, chemical, smallmolecule, etc.) that inhibits a protein that antagonizes HSC survivaland/or proliferation and that is downstream of MYC. Methods ofadministering therapeutically effective amounts of compositions, such asthose containing inhibitors, and determining therapeutic amounts arewell known in the art and described herein. Examples of proteins thatantagonize HSC survival and/or proliferation and that are downstream ofMYC include, without limitation, of pTEN, PP2A, PHLPP, CTMP. Any methodknown in the art for inhibiting protein and/or gene expression,activity, and/or function may be used, including without limitation themethods disclosed herein. Non-limiting examples include geneticinhibitors, small molecule inhibitors, RNA interference, and antibodies.

Activities of Full-Length MYC Proteins

In other embodiments, a MYC-composition of the present disclosurecontains a full-length MYC polypeptide having at least one MYC activity,a fragment of a MYC protein that retains at least one activity of afull-length MYC protein, a homologue of a MYC protein that retains atleast one activity of a full-length MYC protein, or an analogue of a MYCprotein that retains at least one activity of a full-length MYC protein.

Full-length MYC proteins of the present disclosure have numerousactivities. Examples of such activities include, without limitation,transcription factor activity, protein binding activity, nucleic acidbinding activity, cell proliferation regulation activity, cell growthregulation activity, apoptosis regulation activity, morphogenesisregulation activity, development regulation activity, and enhancedhematopoietic compartment reconstitution activity.

In some embodiments a MYC-composition of the present disclosure has aMYC activity that enhances hematopoietic compartment reconstitution inan HSC transplant recipient, including, without limitation, a bonemarrow transplant recipient. Without wishing to be bound by theory, itis believed that MYC activity enhances productive homing of thetransplanted HSCs to bone marrow niches in the recipient. The recoverytime of mature hematopoietic lineages following HSC transplantation islargely dependent of the ability of the administered HSCs to home tobone marrow niches. Once the HSCs arrive at the bone marrow niches, theyneed to establish a molecular crosstalk with the niche-resident cells.This crosstalk is thought to regulate the nature and levels ofcell-intrinsic signals within the HSCs that regulate their survival,proliferation, self-renewal, and differentiation. While the full extentof the surface molecules that regulate the homing and polarized celldivision process between bone marrow niches and HSCs is not yet fullyunderstood, it is clear that several adhesion molecules, such asP-selectin and E-selectin, are involved in this process. Without wishingto be bound by theory, it is believed that MYC is a key regulator ofexpression of P-selectin and E-selectin, in addition to PSGL1, VLA4,VLA5, LFA1, and CD26 in the homing and maintenance of HSCs at bonemarrow niches. It is also believed that MYC is a regulator of survivaland differentiation signals within the HSCs. Further, it is believedthat MYC regulates the expression and/or function of several additionalpathways, such as the Wnt pathway, that are involved in the maintenanceof HSC pluripotency and self-renewal. This molecular interplay enablesthe HSCs to resume a near-quiescent state that supports their asymmetriccell division, allowing for the generation of short-term HSCs that giverise to mature hematopoietic lineages, and for a long-term HSCcompartment that enables the successful reconstitution of thehematopoietic lineages for the lifetime of the HSC transplant recipient.Thus, administering a MYC-composition of the present disclosure to anHSC transplant recipient results in an at least a 50% increase in HSCproductive homing to bone marrow niches in the recipient.

In further embodiments, a MYC-composition of the present disclosurecontains a MYC polypeptide whose activity enhances hematopoieticcompartment autoreconstitution in a subject in need of hematopoieticcompartment autoreconstitution. Without wishing to be bound by theory,it is believed that MYC activity enhances the ability of endogenous HSCsto self-renew and to differentiate into all hematopoietic compartmentlineages, thus enhancing hematopoietic compartment autoreconstitution.Without wishing to be bound by theory, it is also believed that MYCactivity enhances HSC homing to their bone marrow niches.

Advantageously, administering MYC in the form of a MYC-compositionresults in transient MYC activity in a subject. This transient MYCactivity avoids the potentially negative effects of prolonged MYCactivity, such as oncogenicity.

Additionally, MYC-compositions of the present disclosure can increasethe intracellular levels of MYC in both endogenous and exogenouslytransplanted HSCs and committed lineage precursors. Thus, in certainembodiments, administering a MYC-composition of the present disclosureto a subject in need of hematopoietic compartment autoreconstitutionresults in an expansion of endogenous HSCs. In other embodiments,administering a MYC-composition of the present disclosure to an HSCtransplant recipient, such as a bone marrow transplant recipient,results in an expansion of the transplanted HSCs.

As disclosed herein, a therapeutically effective amount aMYC-composition of the present disclosure is at least about or about 0.5μ/ml to at least about or about 100 μ/ml, for example at least about orabout 0.5 μ/ml, at least about or about 0.6 μ/ml, at least about orabout 0.7 μ/ml, at least about or about 0.8 μ/ml, at least about orabout 0.9 μ/ml, at least about or about 1 μ/ml, at least about or about2 μ/ml, at least about or about 3 μ/ml, at least about or about 4 μ/ml,at least about or about 5 μ/ml, at least about or about 6 μ/ml, at least7 about or about μ/ml, at least about or about 8 μ/ml, at least about orabout 9 μ/ml, at least about or about 10 μ/ml, at least about or about15 μ/ml, at least about or about 20 μ/ml, at least about or about 25μ/ml, at least about or about 30 μ/ml, at least about or about 35 μ/ml,at least about or about 40 μ/ml, at least about or about 45 μ/ml, atleast about or about 50 μ/ml, at least about or about 55 μ/ml, at leastabout or about 60 μ/ml, at least about or about 65 μ/ml, at least aboutor about 70 μ/ml, at least about or about 75 μ/ml, at least about orabout 80 μ/ml, at least about or about 85 μ/ml, at least about or about90 μ/ml, at least about or about 95 μ/ml, or at least about or about 100μ/ml of the MYC-composition.

Bcl-2-Compositions

Certain aspects of the present disclosure relate to treating apopulation of hematopoietic stem cells (HSCs) with a compositioncontaining a Bcl-2-composition to enhance hematopoietic compartmentreconstitution. HSCs of the present disclosure may be treated with theBcl-2-composition alone, or in combination with a MYC-composition of thepresent disclosure. Any method of treating cells with a composition,such as fusion protein, known in the art and disclosed herein may beused. For example, a population of hematopoietic stem cells may becultured in the presence of the Bcl-2-composition. In some embodiments,a population of hematopoietic stem cells may be treated with acombination of a MYC composition of the present disclosure along with aBcl-2-composition of the present disclosure. Other aspect of the presentdisclosure relate to administering to a subject in need thereof, acomposition containing a Bcl-2-composition to enhance hematopoieticcompartment formation in the subject.

In some embodiments, a composition containing a Bcl-2-composition of thepresent disclosure may be administered to a subject in need thereof, toenhance hematopoietic compartment formation in the subject. Withoutwishing to be bound by theory, it is believed that the Bcl-2-compositionmay keep more exogenous and/or endogenous hematopoietic stem cells alive(i.e., increase cell survival) long enough to enhance hematopoietic cellcompartment formation (e.g., hematopoietic compartment reconstitutionand/or engraftment by exogenous HSCs or autoreconstitution by endogenousHSCs). Exemplary methods of determining hematopoietic cell compartmentformation are disclosed herein and known in the art. TheBcl-2-composition may be administered in addition to or instead of aMYC-composition of the present disclosure.

As used herein, a “Bcl-2-composition” refers to a Bcl-2 polypeptide; avariant or mutant of a Bcl-2 polypeptide, a modified Bcl-2 polypeptide,a homologue of a Bcl-2 polypeptide; an analogue of a Bcl-2 polypeptide;a biologically active fragment of a Bcl-2 polypeptide; a downstreamtarget of a Bcl-2 polypeptide, a homologue thereof, an analogue thereof,or a biologically active fragment thereof; and a fusion proteincontaining a Bcl-2 polypeptide, a homologue thereof, an analoguethereof, and a biologically active fragment thereof. A Bcl-2-compositionof the present disclosure includes any Bcl-2 polypeptide, variantthereof, mutant thereof, homologue thereof, analogue thereof, orbiologically active fragment thereof known in the art (e.g., US PatentApplication Publication Nos. US 2007/0116691, US 2010/0047217, and US2010/0279351).

In certain preferred embodiments, Bcl-2-compositions of the presentdisclosure are fusion proteins that contain a Bcl-2 polypeptide, variantthereof, mutant thereof, homologue thereof, analogue thereof, orbiologically active fragment thereof that has been coupled (e.g., fused)to a protein transduction domain (PTD).

Bcl-2 Polypeptides

In some embodiments, a Bcl-2-composition of the present disclosure is aBcl-2 polypeptide. A Bcl-2 polypeptide of the present disclosureincludes, without limitation, any polypeptide having the activity of aBcl-2 protein.

As used herein, “Bcl-2,” “Bcl-2 polypeptide,” and “Bcl-2 protein” areused interchangeably and refer to a protein that is a member of theBcl-2 protein family that has one or more and/or all Bcl-2 homology (BH)domains, such as but not limited to, BH1, BH2, BH3, and BH4. Members ofthe bcl-2 protein family typically form heterodimer or homodimers, andfunction as regulators of apoptosis. In certain preferred embodiments,Bcl-2 polypeptides of the present disclosure have anti-apoptoticactivity.

Bcl-2-compositions of the present disclosure may allow for an increasein Bcl-2 activity in a subject in need of hematopoietic compartment cellformation by the exogenous addition of Bcl-2, without the need foroverexpressing endogenous Bcl-2 or recombinantly expressing Bcl-2 viagenetic manipulation.

Bcl-2 polypeptides of the present disclosure include, withoutlimitation, full length Bcl-2 proteins, fragments that retain theactivity of a full-length Bcl-2 protein, homologues thereof, andanalogues thereof. In some embodiments, Bcl-2 fragments that retain theactivity of a full-length Bcl-2 protein include a truncated form ofBcl-2 that has been deleted for the unstructured loop domain (Anderson,M., et al. (1999). Refolding, purification and characterization of aloop deletion mutant of human Bcl-2 from bacterial inclusion bodies.Prot Expr. Purif. 15, 162-70). Bcl-2 polypeptides of the presentdisclosure may be produced by any suitable method known in the art. Forexample, a Bcl-2 polypeptide may be purified from a native source, maybe recombinantly expressed, or may be chemically synthesized.

Bcl-2 Proteins

Examples of full length Bcl-2 proteins suitable for use in any of themethods of the present disclosure include, without limitation, Bcl-2,Bcl-x, Bcl-XL, Mcl-1, CED-9, Bcl-2 related protein A1, Bfl-1, and Bcl-w.

In certain preferred embodiments, the Bcl-2 polypeptide is a full-lengthhuman Bcl-2 polypeptide that has been deleted for the unstructured loopdomain. The human Bcl-2 polypeptide may have one or more of thefollowing features: the polypeptide may be a polymer of 239 amino acids,the polypeptide may have a molecular weight of approximately 26.3 kDa,or the polypeptide may contain at least one Bcl-2 homology (BH) domain,such as BH1, BH2, BH3, and BH4. Preferably, the human Bcl-2 polypeptideis the Bcl-2 polypeptide having NCBI Accession Number NP_000624.2.Moreover, a Bcl-2 polypeptide of the present disclosure may be a Bcl-2polypeptide that has not undergone any post-translational modifications.Alternatively, a Bcl-2 polypeptide of the present disclosure may be aBcl-2 polypeptide that has undergone post-translational modifications.

In some embodiments, the Bcl-2 polypeptide is a fusion proteincontaining a protein transduction domain (PTD). In certain embodiments,the Bcl-2 polypeptide is a fusion protein containing a TAT protein, orfragment thereof, of the present disclosure. In some embodiments, theBcl-2 polypeptide is a TAT-Bcl-2Δ fusion protein, where the Bcl-2polypeptide has a deletion of the unstructured loop domain. In certainembodiments, the TAT-Bcl-2Δ fusion protein has the following amino acidsequence (SEQ ID NO: 3):

MRKKRRQRRRMAHAGRSGYDNREIVMKYIHYKLSQRATSGISIEAAGPALSPVPPVVHLTLRQAGDDFSRRYRRDFAEMSSQLHLTPFTARGCFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLALVGACITLGAYLSHKKGELNSKLEGKPIPNPLLGLDSTRTGHHHHHH

In the TAT-Bcl-2Δ polypeptide of SEQ ID NO: 3, amino acids 2-10correspond to the HIV TAT protein transduction domain, amino acids11-212 correspond to the amino acid sequence of Bcl-2A, amino acids4213-226 correspond to a 14 amino acid V5 epitope, and amino acids230-235 correspond to a 6 Histidine tag.

Biologically Active Bcl-2 Fragments

In other embodiments, a Bcl-2-composition of the present disclosure is abiologically active fragment of a full-length Bcl-2 protein that retainsat least one activity of a full-length Bcl-2 protein. The Bcl-2polypeptide may be a fragment of Bcl-2, Bcl-x, Bcl-XL, Mcl-1, CED-9,Bcl-2 related protein A1, Bfl-1, or Bcl-w.

A Bcl-2 fragment of the present disclosure may contain at least 10, atleast 15, at least 20, at least 25, at least 30, at least 35, at least40, at least 45, at least 50, at least 55, at least 60, at least 65, atleast 70, at least 75, at least 80, at least 85, at least 90, at least95, at least 100, at least 110, at least 120, at least 130, at least140, at least 150, at least 160, at least 170, at least 180, at least190, at least 200, at least 210, at least 220, at least 230, or moreconsecutive amino acid residues of the amino acid sequence of a Bcl-2protein.

Bcl-2 Homologues and Analogues

In other embodiments, a Bcl-2-composition of the present disclosure is ahomologue or analogue of a Bcl-2 protein or fragment thereof. Forexample, a Bcl-2 polypeptide of the present disclosure may include anamino acid sequence that is at least 40% to 100% identical, e.g., atleast 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%,90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percent from about40% to about 100% identical to a Bcl-2 protein or fragments thereof. Incertain embodiments, the Bcl-2 polypeptide is a homologue or analogue ofBcl-2, Bcl-x, Bcl-XL, Mcl-1, CED-9, Bcl-2 related protein A1, Bfl-1,Bcl-w, or fragments thereof.

Bcl-2 polypeptides of the present disclosure also include functionalhomologues or analogues of the human Bcl-2 polypeptide having NCBIAccession Number NP_00624.2, or a fragment thereof. In certainembodiments, the Bcl-2 homologue or analogue contains an amino acidsequence that is at least 40% to 100% identical, e.g., at least 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%,92%, 94%, 95%, 96%, 97%, 98%, or any other percent from about 40% toabout 100% identical to the Bcl-2 polypeptide sequence of NCBI AccessionNumber NP_00624.2 or fragment thereof.

In other embodiments, the Bcl-2 homologue or analogue contains apolypeptide sequence of at least 10 amino acids, at least 20 aminoacids, at least 30 amino acids, at least 40 amino acids, at least 50amino acids, at least 60 amino acids, at least 70 amino acids, at least80 amino acids, at least 90 amino acids, at least 100 amino acids, atleast 110 amino acids, at least 120 amino acids, at least 130 aminoacids, at least 140 amino acids, at least 150 amino acids, at least 160amino acids, at least 170 amino acids, at least 180 amino acids, atleast 190 amino acids, at least 200 amino acids, at least 210 aminoacids, at least 220 amino acids, at least 230 amino acids, or more inlength that is at least 50% to 100% identical, e.g., at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%,96%, 97%, 98%, or any other percent from about 50% to about 100%identical to the Bcl-2 polypeptide sequence of NCBI Accession NumberNP_00624.2 or fragment thereof.

As disclosed herein, suitable Bcl-2 polypeptides also includeconservatively modified variants of Bcl-2 polypeptides of the presentdisclosure.

Activities of Full-Length Bcl-2 Proteins

In other embodiments, a Bcl-2-composition of the present disclosurecontains a full-length Bcl-2 polypeptide having at least one Bcl-2activity, a fragment of a Bcl-2 protein that retains at least oneactivity of a full-length Bcl-2 protein, a homologue of a Bcl-2 proteinthat retains at least one activity of a full-length Bcl-2 protein, or ananalogue of a Bcl-2 protein that retains at least one activity of afull-length Bcl-2 protein.

Full-length Bcl-2 proteins of the present disclosure have numerousactivities. Examples of such activities include, without limitation,apoptosis regulation activity, cell survival regulation activity,protein binding activity, mitochondrial membrane permeability regulationactivity, caspase regulation activity, voltage-dependent anion channelregulation activity, G2 checkpoint regulation activity, outermitochondrial membrane channel (VDAC) regulation activity, mitochondrialmembrane potential regulation activity, protein channel activity, andcytochrome C regulation activity.

In some embodiments a Bcl-2-composition of the present disclosure has aBcl-2 activity that either alone or in combination with aMYC-composition enhances hematopoietic compartment reconstitution in anHSC transplant recipient, including, without limitation, a bone marrowtransplant recipient, when the HSCs are treated with the compositionsprior to HSC transplantation.

As disclosed herein, a therapeutically effective amount aBcl-2-composition of the present disclosure is at least about or about0.5 μ/ml to at least about or about 100 μ/ml, for example at least aboutor about 0.5 μ/ml, at least about or about 0.6 μ/ml, at least about orabout 0.7 μ/ml, at least about or about 0.8 μ/ml, at least about orabout 0.9 μ/ml, at least about or about 1 μ/ml, at least about or about2 μ/ml, at least about or about 3 μ/ml, at least about or about 4 μ/ml,at least about or about 5 μ/ml, at least about or about 6 μ/ml, at least7 about or about μ/ml, at least about or about 8 μ/ml, at least about orabout 9 μ/ml, at least about or about 10 μ/ml, at least about or about15 μ/ml, at least about or about 20 μ/ml, at least about or about 25μ/ml, at least about or about 30 μ/ml, at least about or about 35 μ/ml,at least about or about 40 μ/ml, at least about or about 45 μ/ml, atleast about or about 50 μ/ml, at least about or about 55 μ/ml, at leastabout or about 60 μ/ml, at least about or about 65 μ/ml, at least aboutor about 70 μ/ml, at least about or about 75 μ/ml, at least about orabout 80 μ/ml, at least about or about 85 μ/ml, at least about or about90 μ/ml, at least about or about 95 μ/ml, or at least about or about 100μ/ml of the Bcl-2-composition.

Protein Transduction Domains

Certain aspects of the present disclosure relate to fusion proteinscontaining a protein transduction domain. In some embodiments aMYC-composition of the present disclosure is a fusion protein containinga protein transduction domain fused to a MYC polypeptide. In otherembodiments, a Bcl-2-composition of the present disclosure is a fusionprotein containing a protein transduction domain fused to a Bcl-2polypeptide.

As used herein, the terms “peptide transduction domain,” “proteintransduction domain,” and “PTD” are used interchangeably and refer to apeptide sequence or domain of a protein that promotes penetration ofprotein into a mammalian cell and/or compartment(s) within a mammaliancell. In one non-limiting example, a PTD promotes penetration of acoupled peptide and/or protein into the nucleus of a cell.

PTDs of the present disclosure may be isolated from a PTD-containingprotein by any method of isolating a protein domain known in the art,such as standard molecular biology and biochemical techniques.Alternatively, PTDs of the present disclosure may be synthesized.Suitable PTDs of the present disclosure may be about 8 to about 30 aminoacid residues in length, and enriched in basic amino acid residues, suchas argentine (Arg) and lysine (Lys).

Suitable PTDs of the present disclosure are not strongly immunogenic,and as such do not induce a strong immune response when administered toa subject. Moreover, PTDs of the present disclosure also do not inducean immune response when administered to an immunocompromised subject,such a subject who has received, is receiving, or will receive a bonemarrow or HSC transplant.

As disclosed herein, PTDs of the present disclosure are coupled (e.g.,fused, conjugated, cross-linked, etc.) to a peptide and/or protein inorder to facilitate the penetration of the peptide and/or protein into amammalian cell and/or compartment within a mammalian cell. For example,in certain embodiments a PTD of the present disclosure is coupled to aMYC protein.

Protein transduction domains suitable for use in any of the methods ofthe present disclosure include any PTD known in the art (e.g., U.S.Patent Application Publication Nos. US 2007/0116691 and US2010/0055129). For example, suitable PTDs may be obtained or derivedfrom proteins that include, without limitation, lentiviral TAT(Trans-Activator of Transcription) proteins, lentiviral VPR proteins,herpesviral VP22 proteins, and homeoproteins.

Examples of suitable PTDs obtained or derived from lentiviral TATproteins include, without limitation, the PTD from a TAT protein of aTAT protein-containing virus, the PTD from a TAT protein of a TATprotein-containing lentivirus, the PTD from the HIV-1 TAT protein, thePTD from the HIV-2 TAT protein, the PTD from the SIV TAT protein, thePTD from a primate lentivirus TAT protein, the PTD from an ovinelentivirus TAT protein, the PTD from a bovine lentivirus TAT protein,the PTD from an equine lentivirus TAT protein, the PTD from a felinelentivirus TAT protein, a PTD from the TAT protein of a subvariant ofHIV, SIV, primate lentivirus, ovine lentivirus, bovine lentivirus,equine lentivirus, or feline lentivirus, and homologs thereof. Incertain embodiments, the PTD is amino acid residues 48-57 of the HIV TATprotein (TAT_([48-57])). In other embodiments, the PTD is amino acidresidues 57-48 of the HIV TAT protein (TAT_([57-48])).

Examples of suitable PTDs that may obtained or derived from lentiviralVPR proteins include, without limitation, the PTD from a VPR protein ofa VPR protein-containing virus, the PTD from a VPR protein of a VPRprotein-containing lentivirus, the PTD from the HIV-1 VPR protein, thePTD from the HIV-2 VPR protein, the PTD from the SIV VPR protein, thePTD from a primate lentivirus VPR protein, the PTD from an ovinelentivirus VPR protein, the PTD from a bovine lentivirus VPR protein,the PTD from an equine lentivirus VPR protein, the PTD from a felinelentivirus VPR protein, a PTD from the VPR protein of a subvariant ofHIV, SIV, primate lentivirus, ovine lentivirus, bovine lentivirus,equine lentivirus, or feline lentivirus, and homologs thereof.

Examples of suitable PTDs that may obtained or derived from herpesviralVP22 proteins include, without limitation, the PTD from the humanherpesvirus 1(HSV-1) VP22 protein, the PTD from the human herpesvirus 2(HSV-2) VP22 protein, the PTD from the BHV-1 VP22 protein, the PTD fromthe Psittacid herpesvirus 1VP22 protein, the PTD from the Equineherpesvirus 1 VP22 protein, the PTD from the Equine herpesvirus 4 VP22protein, the PTD from the Gallid herpesvirus 2 VP22 protein, the PTDfrom the Varicella-zoster virus VP22 protein, and homologs thereof.

Examples of suitable PTDs that may be obtained or derived fromhomeodomain transcription factors include, without limitation, thehomeodomain (HD) from the Drosophila Antennapedia (Antp) protein, the HDfrom the Drosophila Fushi tarazu (Ftz) protein, the HD from theDrosophila Engrailed (En) protein, the HD from the chick Engrailed-2protein, the HD from mammalian homeoproteins, the HD from humanhomeoproteins, the HD from human Hox-A5 homeoprotein, the HD from humanHox-A4 homeoprotein, the HD from human Hox-B5 homeoprotein, the HD fromhuman Hox-B6 homeoprotein, the HD from human Hox-B7 homeoprotein, the HDfrom human HOX-D3 homeoprotein, the HD from human GOX homeoprotein, theHD from human MOX-2 homeoprotein, the HD from human Hoxc-8 homeoprotein,the HD from human Islet-1 (Is1-1) homeoprotein, and homologs thereof.

Additionally, suitable PTDs include, without limitation, the PTD derivedfrom Kaposi-FGF (K-FGF or FGF-4), the PTD derived from FGF-2, the PTDderived from FGF-1, and the PTD from other members of the FGF-family ofproteins.

Other suitable PTDs include synthetic PTDs (e.g., Beerens, AMJ et al.Curr Gene Ther. 2003 October; 3(5):486-94).

Further suitable PTDs include, without limitation, a CHARIOT™ peptide(Active Motif, Carlsbad, Calif.).

In some embodiments, PTDs of the present disclosure are producedrecombinantly, while in others the PTDs are produced synthetically orare purified from a native source.

PTD Fusion Protein Modifications

In some embodiments, PTD-containing fusion proteins of the presentdisclosure include PTD-MYC fusion proteins and PTD-Bcl-2 fusion proteinsthat contain one or more molecules that link the PTD to the MYC or Bcl-2polypeptide. In some embodiments, the one or more linker molecules areamino acid peptides.

PTD-containing fusion proteins of the present disclosure may furthercontain at least one amino acid sequence that facilitates purificationof the fusion proteins. For example, the PTD-MYC fusion proteins maycontain a protein tag, such as a polyhistidine tag, such as a sixhistidine epitope tag. Alternatively, the PTD-containing fusion proteinsmay containa V5 domain. Accordingly, in certain embodiments,PTD-containing fusion proteins of the present disclosure further containa polyhistidine tag. Preferably, the polyhistidine tag is a 6-histidinetag. More preferably, the histidine tag contains the sequence HHHHHH.Additionally, the histidine tag may be added to a PTD-containing fusionprotein of the present disclosure by any suitable method known in theart. For example, a sequence may be cloned into an expression vectorencoding a polyhistidine tag. Alternatively, a polyhistidine tag may beadded by PCR (i.e., the PCR primers contain a polyhistidine sequence).

Moreover, a PTD-containing fusion protein of the present disclosure mayalso contain at least one protein tag. In some embodiments, the at leastone protein tag is an epitope tag. Preferably, the epitope tag is a V5epitope tag. In some embodiments, the V5 epitope tag contains the aminoacid sequence: GKPIPNPLLGLDST, while in other the V5 epitope tagcontains the amino acid sequence: IPNPLLGLD. The amino acids may beeither in the D formation, or in the L formation. In some embodiments, afirst plurality of amino acids is in the D formation and a secondplurality is in the L formation. Additionally, aV5 epitope tag of thepresent disclosure may be added to a PTD-containing fusion protein ofthe present disclosure by any suitable method known in the art. Forexample, a PTD-containing fusion protein sequence may be cloned into anexpression vector encoding a V5 epitope tag. Alternatively, aV5 epitopetag may be added by PCR (i.e., the PCR primers contain a V5 epitopesequence).

In certain embodiments, a PTD-containing fusion protein of the presentdisclosure further contains a polyhistidine tag and an epitope tag.Preferably, the PTD-containing fusion protein contains a 6-histidine tagand a V5 epitope tag.

In some embodiments, a PTD-containing fusion protein of the presentdisclosure can be arranged in any desired order. In some embodiments,the PTD-containing fusion protein can be arranged in order of a) theprotein transduction domain connected in frame to the Myc or Bcl-2polypeptide, b) the Mycor Bcl-2 polypeptide connected in frame to the V5domain, and c) the V5 domain connected in frame to the six histidineepitope tag. In some embodiments, the PTD-containing fusion protein hasan order of components of a) the Myc or Bcl-2 polypeptide connected inframe to the protein transduction domain, b) the protein transductiondomain connected in frame to the V5 domain, and c) the V5 domainconnected in frame to the six histidine epitope tag. In someembodiments, additional intervening amino acid sequences can be includedbetween each of the sequences. In some embodiments, additional aminoacid sequences can be included at the start and/or end of the sequences.

In some embodiments, the PTD-containing fusion protein contains aprotein transduction domain, a Myc or Bcl-2 polypeptide, and a shortpeptide domain. The short peptide domain can be varied. In someembodiments, the short peptide domain is selected from at least one of aV5, a histidine-tag, HA (hemagglutinin) tags, FLAG tag, CBP (calmodulinbinding peptide), CYD (covalent yet dissociable NorpD peptide), StrepII,or HPC (heavy chain of protein C). In some embodiments, the shortpeptide domain is about 10 or 20 amino acids long. In some embodiments,the short peptide domain is 2-20, for example 6-20 amino acids inlength. In some embodiments, two of the above listed items (for example,V5 and the his-tag) can be used together as the short peptide domain.

Construction of PTD-Containing Fusion Proteins

PTD-containing fusion proteins of the present disclosure may beconstructed by any suitable method known in the art (e.g., U.S. PatentApplication Publication No. US 2010/0055129).

In one non-limiting example, a nucleic acid sequence encoding a PTD-MYCfusion protein of the present disclosure may be generated by PCR. Thismay be accomplished by designing a forward primer for a MYC sequencethat contains an in frame PTD sequence, such as the RKKRRQRRR9-amino-acid sequence of TAT, and a reverse primer for the MYC sequencethat is designed to remove the stop codon. The PCR product from a PCRreaction using such primers may then be cloned into any suitableexpression vector known in the art.

In one non-limiting example, a nucleic acid sequence encoding aPTD-Bcl-2 fusion protein of the present disclosure may be generated byPCR. This may be accomplished by designing a forward primer for a Bcl-2sequence that contains an in frame PTD sequence, such as the RKKRRQRRR9-amino-acid sequence of TAT, and a reverse primer for the Bcl-2sequence that is designed to remove the stop codon. The PCR product froma PCR reaction using such primers may then be cloned into any suitableexpression vector known in the art. The Bcl-2 unstructured loop may beremoved from the BCL-2 coding sequence using a site directed mutagenesiskit.

PTD-Containing Compositions

In other embodiments, PTD-containing fusion proteins of the presentdisclosure are included in a composition. For therapeutic methods, suchfusion protein-containing compositions may include a pharmaceuticallyacceptable carrier, which includes pharmaceutically acceptableexcipients and/or delivery vehicles, for delivering a PTD-containingfusion protein to a subject, such as an HSC transplant recipient.

In some embodiments, the PTD-containing fusion protein is a PTD-MYC, anda therapeutically effective amount of the PTD-MYC fusionprotein-containing composition (PTD-MYC composition) is administered toa subject to achieve an at least 50% acceleration in hematopoieticcompartment reconstitution compared to hematopoietic compartmentreconstitution in a subject that is not administered the secondcomposition. In other embodiments, a therapeutically effective amount ofa PTD-MYC composition is administered to a subject to achieve enhancedhematopoietic compartment autoreconstitution compared to hematopoieticcompartment autoreconstitution in a subject that is not administered thePTD-MYC composition. Advantageously, PTD-MYC compositions of the presentdisclosure have low toxicity when administered to a subject.Accordingly, a PTD-MYC composition may be administered in an amount thatranges from about 0.1 to about 50 mg/kg of the weight of the subject. Incertain embodiments, a therapeutically effective amount of a compositioncontaining a PTD-MYC fusion protein is at least 0.1 mg/kg, at least 0.2mg/kg, at least 0.3 mg/kg, at least 0.4 mg/kg, at least 0.5 mg/kg, atleast 0.6 mg/kg, at least 0.7 mg/kg, at least 0.8 mg/kg, at least 0.9mg/kg, at least 1 mg/kg, at least 2 mg/kg, at least 3 mg/kg, at least 4mg/kg, at least 5 mg/kg, at least 6 mg/kg, at least 7 mg/kg, at least 8mg/kg, at least 9 mg/kg, at least 10 mg/kg, at least 20 mg/kg, at least30 mg/kg, at least 40 mg/kg, or at least 50 mg/kg of the weight of thesubject.

In other embodiments, the PTD-containing fusion protein is a PTD-Bcl-2,and a therapeutically effective amount of the PTD-Bcl-2 fusionprotein-containing composition (PTD-Bcl-2-composition) is administeredto a subject to achieve an at least 50% acceleration in hematopoieticcompartment reconstitution compared to hematopoietic compartmentreconstitution in a subject that is not administered the secondcomposition. In other embodiments, a therapeutically effective amount ofa PTD-Bcl-2-composition is administered to a subject to achieve enhancedhematopoietic compartment autoreconstitution compared to hematopoieticcompartment autoreconstitution in a subject that is not administered thePTD-Bcl-2-composition. Advantageously, PTD-Bcl-2-compositions of thepresent disclosure have low toxicity when administered to a subject.Accordingly, a PTD-Bcl-2-composition may be administered in an amountthat ranges from about 0.1 to about 50 mg/kg of the weight of thesubject. In certain embodiments, a therapeutically effective amount of acomposition containing a PTD-Bcl-2 fusion protein is at least 0.1 mg/kg,at least 0.2 mg/kg, at least 0.3 mg/kg, at least 0.4 mg/kg, at least 0.5mg/kg, at least 0.6 mg/kg, at least 0.7 mg/kg, at least 0.8 mg/kg, atleast 0.9 mg/kg, at least 1 mg/kg, at least 2 mg/kg, at least 3 mg/kg,at least 4 mg/kg, at least 5 mg/kg, at least 6 mg/kg, at least 7 mg/kg,at least 8 mg/kg, at least 9 mg/kg, at least 10 mg/kg, at least 20mg/kg, at least 30 mg/kg, at least 40 mg/kg, or at least 50 mg/kg of theweight of the subject.

PTD-MYC compositions of the present disclosure and/orPTD-Bcl-2-compositions of the present disclosure can be administered atleast once a day, at least twice a day, at least three times a day, atleast four times a day, at least five times a day, or more times a day.Alternatively, PTD-MYC compositions of the present disclosure and/orPTD-Bcl-2-compositions of the present disclosure can be administered atleast once every two days, at least once every three days, at least onceevery four days, at least once every five days, or at least once everysix days, at least once every week, at least once every two weeks, atleast once every three weeks, or at least once every four weeks.

Advantageously, PTD-MYC compositions of the present disclosure and/orPTD-Bcl-2-compositions of the present disclosure can acceleratehematopoietic compartment reconstitution from HSC transplantation (e.g.,HSC engraftment), such as from bone marrow transplantation, in a subjectwhen administered from at least about or about 5 days to at least aboutor about 12 hours before an HSC transplant, concurrently with an HSCtransplant, or from at least about or about 12 hours to at least aboutor about 3 weeks after an HSC transplant. A PTD-MYC composition of thepresent disclosure and/or a PTD-Bcl-2-composition of the presentdisclosure may be administered at any suitable location, including,without limitation, the same location where the HSC transplantationoccurs, a clinic or doctor's office that is separate from the locationwhere the HSC transplantation occurs, and the home or residence of thesubject receiving the HSC transplantation. Any method of administeringPTD-MYC compositions of the present disclosure and/orPTD-Bcl-2-compositions of the present disclosure known in the art anddisclosed herein may be used. In some embodiments, the PTD-MYCcomposition and/or the PTD-Bcl-2-composition is administered togetherwith any suitable pharmaceutically acceptable carrier known in the artand disclosed herein. In other embodiments, the PTD-MYC compositionand/or the PTD-Bcl-2-composition is administered as a suitableformulation known in the art, including, without limitation, anintravenous formulation.

As used herein, administering a PTD-MYC composition of the presentdisclosure and/or a PTD-Bcl-2-composition of the present disclosureoccurs “concurrently” with an HSC transplantation when it isadministered either in the same mixture and/or formulation as the HSCsto be transplanted, or in a separate mixture and/or formulation as thatof the HSCs to be transplanted. When the PTD-MYC composition and/or thePTD-Bcl-2-composition is administered in a separate mixture and/orformulation, administering a PTD-MYC composition of the presentdisclosure occurs and/or a PTD-Bcl-2-composition of the presentdisclosure “concurrently” with an HSC transplantation includes, withoutlimitation, administering the PTD-MYC composition and/or thePTD-Bcl-2-composition from at least about or about 11 hours to at leastabout or about 1 minute before HSC transplantation, or from at leastabout or about 1 minute to at least about or about 11 hours after HSCtransplantation. As used herein, administering a PTD-MYC composition ofthe present disclosure and/or a PTD-Bcl-2-composition of the presentdisclosure occurs “before” an HSC transplantation when it isadministered in a separate mixture and/or formulation as that of theHSCs to be transplanted from at least about or about 5 days to at leastabout or about 12 hours before the HSC transplantation. As used herein,administering a PTD-MYC composition of the present disclosure occursand/or a PTD-Bcl-2-composition of the present disclosure “after” an HSCtransplantation when it is administered in a separate mixture and/orformulation as that of the HSCs to be transplanted from at least aboutor about 12 hours to at least about or about 3 weeks after the HSCtransplantation.

Accordingly, in certain embodiments, a PTD-MYC composition of thepresent disclosure and/or a PTD-Bcl-2-composition of the presentdisclosure is administered before, after, or concurrently with an HSCtransplant in a subject. In some embodiments, the PTD-MYC compositionand/or the PTD-Bcl-2-composition is administered at least about or about5 days, at least about or about 4 days, at least about or about 3 days,at least about or about 2 days, at least about or about 1 day, at leastabout or about 23 hours, at least about or about 22 hours, at leastabout or about 21 hours, at least about or about 20 hours, at leastabout or about 19 hours, at least about or about 18 hours, at leastabout or about 17 hours, at least about or about 16 hours, at leastabout or about 15 hours, at least about or about 14 hours, at leastabout or about 13 hours, or at least about or about 12 hours before anHSC transplant. In other embodiments, the PTD-MYC composition and/or thePTD-Bcl-2-composition is administered concurrently with an HSCtransplant. In further embodiments, the PTD-MYC composition and/or thePTD-Bcl-2-composition is administered at least about or about 12 hours,at least about or about 13 hours, at least about or about 14 hours, atleast about or about 15 hours, at least about or about 16 hours, atleast about or about 17 hours, at least about or about 18 hours, atleast about or about 19 hours, at least about or about 20 hours, atleast about or about 21 hours, at least about or about 22 hours, or atleast about or about 23 hours, at least about or about 1 day, at leastabout or about 2 days, at least about or about three days, at leastabout or about 4 days, at least about or about 5 days, at least about orabout 6 days, at least about or about 1 week, at least about or about 2weeks, or at least about or about 3 weeks after an HSC transplant.

Therapeutic Uses

PTD-MYC compositions of the present disclosure and/orPTD-Bcl-2-compositions of the present disclosure find many therapeuticuses in the treatment of various conditions, diseases, and syndromes.Such uses include, without limitation, accelerating hematopoieticcompartment reconstitution in an HSC transplant recipient, such as abone marrow transplant recipient; enhancing hematopoietic compartmentautoreconstitution; treating decreases in the hematopoietic compartmentdue to chemotherapy or radiation therapy; treating bone marrow failuresyndromes; and treating long-term HSC engraftment failure.

As used herein, a “preventing a decrease in hematopoietic compartmentcells” due to chemotherapy, radiation therapy, bone marrow failure,and/or any other condition that leads to loss of hematopoieticcompartment cells, refers to reducing and/or inhibiting the reduction inthe amount of any hematopoietic compartment cells of the presentdisclosure that are lost due to cell necrosis, apoptosis, or the like asa result of the chemotherapy, radiation therapy, bone marrow failure,and/or condition that leads to loss of hematopoietic compartment cells.Any method known in the art and disclosed herein for quantifying cellloss (e.g., quantifying cell necrosis, apoptosis, etc.) may be used. Inone non-limiting example DNA-intercalating dyes are used to quantifycell loss. In some embodiments, “preventing a decrease in hematopoieticcompartment cells” may include, without limitation, an at least about orabout 25% to an at least about or about 99%, or more reduction inhematopoietic compartment cell loss due to chemotherapy, radiationtherapy, bone marrow failure, and/or condition that leads to loss ofhematopoietic compartment cells.

In certain embodiments, PTD-MYC compositions of the present disclosureand/or PTD-Bcl-2-compositions of the present disclosure may be used toreduce the risk of engraftment failure and opportunistic infections inan HSC transplant recipient by accelerating hematopoietic compartmentreconstitution in the recipient.

Alternatively, PTD-MYC compositions of the present disclosure and/orPTD-Bcl-2-compositions of the present disclosure may be used to treatbone marrow failure syndromes in a subject. Bone marrow failuresyndromes can be inherited, or can occur as a result of, for example, aninfectious disease, or chronic fatigue syndrome. Additionally, bonemarrow failure in an HSC transplant recipient can occur as result of,for example, a transplant of an insufficient number of HSCs, atransplant of mismatched HSCs, or the health status of the recipient.

Acquired bone marrow failure syndromes may include, without limitation,aplastic anemia and Gulf War Syndrome.

Inherited bone marrow failure syndromes (IBMFS) may include, withoutlimitation, amegakaryocytic thrombocytopenia, Diamond-Blackfan anemia,dyskeratosis congenita, fanconi anemia, Pearson syndrome, severecongenital neutropenia, Shwachman-Diamond syndrome, and thrombocytopeniaabsent radii, IVIC syndrome, WT syndrome, radio-ulnar synostosis, andataxia pancytopenia.

In further embodiments, PTD-MYC compositions of the present disclosureand/or PTD-Bcl-2-compositions of the present disclosure may be used intreating an HSC transplant recipient whose hematopoietic chimaerism isfailing after an initial engraftment of transplanted HSCs (i.e., failureof long-term HSC engraftment).

Exposure to toxins, such as chemicals, chemotherapeutic drugs, orradiation may lead to a decrease or loss in a subject's hematopoieticcompartment. As used herein, a decrease in the hematopoietic compartmentmay occur when there is a decrease in the total amount cells in thehematopoietic compartment or a decrease in a specific population orsub-population of cells in the hematopoietic compartment. Examples ofspecific populations or sub-populations of cells in the hematopoieticcompartment include, without limitation, myeloid cells, monocytes,macrophages, neutrophils, basophils, eosinophils, erythrocytes,megakaryocytes, platelets, dendritic cells, lymphocytes, T-cellprogenitors, pro-T-cells, pre-T-cells, double positive T-cells, immatureT-cells, mature T-cells, B-cell progenitors, pro-B-cells, earlypro-B-cells, late-pro B-cells, large pre-B-cells, small pre-B-cells,immature B-cells, mature B-cells, NKT-cells, and NK-cells.

Accordingly, in certain embodiments, PTD-MYC compositions of the presentdisclosure and/or PTD-Bcl-2-compositions of the present disclosure maybe used to treat subjects that have been exposed to such toxins. In someembodiments, treating subjects with a PTD-MYC composition of the presentdisclosure and/or a PTD-Bcl-2-composition of the present disclosureprevents a decrease or loss in hematopoietic compartment cells insubjects that are undergoing or have undergone chemotherapy or radiationtherapy. In other embodiments, treating subjects with a PTD-MYCcomposition of the present disclosure and/or a PTD-Bcl-2-composition ofthe present disclosure prevents a decrease or loss in the amount ofendogenous HSCs in subjects that are undergoing or have undergonechemotherapy or radiation therapy.

Hematopoietic Stem Cells

Other aspects of the present disclosure relate to treating a populationof hematopoietic stem cells (HSCs) with a MYC-composition of the presentdisclosure, a Bcl-2-composition of the present disclosure, or both priorto transplanting the HSCs in a subject in need thereof to enhancehematopoietic compartment reconstitution. Other aspects of the presentdisclosure relate to enhancing hematopoietic compartment cell formationin a subject in need thereof by administering a MYC-composition of thepresent disclosure, a Bcl-2-composition of the present disclosure, orboth to induce HSCs to generate hematopoietic compartment cells. A“subject”, “patient”, or “host” to be treated by the methods of thepresent disclosure may be a human or non-human such as any of thenon-human animal disclosed herein, in need of hematopoietic compartmentcell formation (e.g., hematopoietic compartment reconstitution orautoreconstitution) enhancement. As disclosed herein HSCs of the presentdisclosure pre-treated with a MYC-composition of the present disclosure,a Bcl-2-composition of the present disclosure, or both may be washedprior to being administered to a subject in need of hematopoieticcompartment reconstitution. Alternatively, the pre-treated HSCs may beadministered to the subject without being washed prior to administrationas the transient effects of the MYC-composition and/or Bcl-2-compositionare not harmful to the subject. Any suitable solution known in the artas disclosed herein may be used to wash the pre-treated HSCs. In onenon-limiting example, a pH-balanced saline solution is used to wash thepre-treated HSCs prior to administering the HSCs to the subject. Thepre-treated HSCs may be washed from less than about or about 1 minute toabout 1 hour, about 4 hours, about 6 hours, about 8 hours, about 12hours, about 24 hours or more prior administering to the subject.

HSCs of the present disclosure are able to give rise to all cell typesof the hematopoietic compartment, including, without limitation themyeloid lineage, which includes, without limitation, monocytes,macrophages, neutrophils, basophils, eosinophils, erythrocytes,megakaryocytes, platelets, and dendritic cells; and the lymphoidlineage, which includes, without limitation, T-cells, B-cells,NKT-cells, and NK cells.

In some embodiments, a MYC-composition of the present disclosureaccelerates hematopoietic compartment reconstitution in HSC transplantrecipients. As disclosed herein, HSC transplant recipients may receive abone marrow transplant, an HSC-enriched bone marrow transplant, atransplant of cord blood, a transplant of HSC-enriched cord blood, atransplant of placenta-derived blood, a transplant of purified orpartially-purified HSCs, a transplant of HSCs derived from an HSC cellline, or a transplant of conditionally immortalized HSCs. In suchembodiments, HSCs may be administered as a step in the process of an HSCtransplantation procedure. As disclosed herein, the HSCs may beincluded, without limitation, in transplanted bone marrow, transplantedcord blood, or in transplanted cell lines. In certain preferredembodiments, the transplant recipient is a human subject. Suitable HSCsmay be obtained by any suitable technique known in the art. For example,HSCs may be found in the bone marrow of a donor, which includes femurs,hip, ribs, sternum, and other bones. Any method known in the art forextracting or harvesting bone marrow cells may be used. In onenon-limiting example, HSCs may be obtained directly from the marrowcavity of the hip using a needle and syringe to aspirate cells from themarrow cavity. Rich marrow may be obtained from the hip by performingmultiple small aspirations.

HSCs suitable for use with the methods of the present disclosure may beproduced from embryonic stem (ES) cells and/or induced pluripotent stem(iPS) cells. Any method of producing HSCs from ES cells and/or iPS cellsknown in the art may be used (e.g., Keller, G. Genes Dev. 200519:1129-1155; and Papapetrou Sadelain, F1000 Med Rep. 2010 Jun. 16; 2).For example, HSCs may be produced from ES cells by patterning thehematopoietic development of ES cell culture on the hematopoieticcommitment in the early embryo (e.g., Keller, G. Genes Dev. 200519:1129-1155).

Additionally, HSCs suitable for use with the methods of the presentdisclosure may be obtained by any suitable technique known in the art.For example, HSCs may be found in the bone marrow of a donor, whichincludes femurs, hip, ribs, sternum, and other bones. Any method knownin the art for extracting or harvesting bone marrow cells may be used.In one non-limiting example, HSCs may be obtained directly from themarrow cavity of the hip using a needle and syringe to aspirate cellsfrom the marrow cavity. Rich marrow may be obtained from the hip byperforming multiple small aspirations.

Suitable HSCs may also be obtained from peripheral blood cells found inthe blood of a donor, often following pre-treatment with cytokines, suchas G-CSF (granulocyte colony-stimulating factors), that induce HSCs tobe released from the bone marrow compartment of the donor. HSCs may alsobe obtained from peripheral blood that has undergone an apheresisprocedure to enrich for HSC. Any apheresis procedure known in the artmay be used. In certain embodiments, the apheresis procedure is aleukapheresis procedure.

Additionally, suitable HSCs may be obtained from umbilical cord blood,placenta, and mobilized peripheral blood. For experimental purposes,fetal liver, fetal spleen, and AGM (Aorta-gonad-mesonephros) of animalsare also useful sources of HSCs. Additionally, HSCs may be procured froma source that obtained HSCs from the bone marrow, peripheral blood,umbilical cord, or fetal tissue of a donor. Alternatively, the HSCs maybe included in a bone marrow, peripheral blood, umbilical cord, or fetaltissue sample of a donor.

In some embodiments, HSCs are obtained from a human umbilical cord orplacenta. Another source of HSCs that may be utilized is the developingblood-producing tissues of fetal animals. In humans, HSCs may be foundin the circulating blood of a human fetus by about 12 to 18 weeks. Insome embodiments, human HSCs are obtained from any source, e.g., thebone marrow, umbilical cord, peripheral blood, or fetal tissue of blood,of type A+, A−, B+, B−, O+, O−, AB+, and AB-donors. In otherembodiments, human HSCs are obtained from any source, e.g., the bonemarrow, umbilical cord, peripheral blood, or fetal tissue of blood, ofuniversal donors or donors having a rare blood type. Rare blood typesare known in the art and include, without limitation, Oh, CDE/CDE,CdE/CdE, C^(w)D−/C^(w)D−, −D−/−D−, R_(null), Rh:−51, LW(a−b+), LW(a−b−),−S−s−U−, S−s−U(+), pp, Pk, Lu(a+b−), Lu(a−b−), Kp(a+b−), Kp(a−b−),Js(a+b−), Ko, K:−11, Fy(a−b−), Jk(a−b−), Di(b−), I−, Yt(a−), Sc:−1,Co(a−), Co(a−b−), Do(a−), Vel−, Ge−, Lan−, Lan(+), Gy(a−), Hy−, At(a−),Jr(a−), In(b−), Tc(a−), Cr(a−), Er(a−), Ok(a−), JMH−, and En(a−).

In other embodiments, human HSCs are obtained from any source, e.g., thebone marrow, umbilical cord, peripheral blood, or fetal tissue of blood,of donors having an auto-immune disorder, immune deficiency, or anyother disease or disorder that would benefit from a transplantation ofHSCs. Such donors may also be the recipients. Advantageously, HSCsobtained from such donor may be used for personalized HSC therapy.

In one non-limiting example, human HSCs may be obtained by anesthetizingthe stem cell donor, puncturing the posterior superior iliac crest witha needle, and performing aspiration of bone marrow cells with a syringe.In another non-limiting example, HSCs may be obtained from theperipheral blood of a donor, where a few days prior to harvesting thestem cells form the peripheral blood, the donor is injected with G-CSFin order to mobilize the stem cells to the peripheral blood.

Accordingly, in some embodiments, HSCs are obtained from an autologousdonor, that is the donor will also be the recipient of the HSCs derivedfrom such HSCs. Any methods known in the art and described herein may beused to obtain HSCs from the autologous donor. The HSCs and/or anytherapeutic products derived or produced therefrom are thentransplanted, administered, and or transfused back to the originaldonor. Similarly, HSCs may be obtained from an allogenic donor, such asa sibling, parent, or other relative of a subject in need of an HSCtransplantation. In one non-limiting example, allogenic HSCs areobtained by collecting HSCs from different blood groups or majorhistocompatibility complex (MHC) or human leukocyte antigen (HLA)matching sources. Autologous and/or allogenic HSC transplantation mayoccur at any time after the donation, such as days later, months later,or even years later. Autologous donation may be particularly useful incases where the subject in need of HSCs would have a negative,deleterious, or toxic reaction to transplantation and/or transfusion ofHSCs from any other donor, including allogenic and/or universal donors.Examples of patients that may benefit from autologous and/or allogenicdonation are well known in the art and include, without limitation,those suffering from an autoimmune disorder, blood disease or disorder,immune disease or disorder, or other related diseases or conditions.

Cells obtained from, for example, bone marrow, peripheral blood, or cordblood, are typically processed after extraction or harvest. Any methodknown in the art for processing extracted or harvest cells may be used.Examples of processing steps include, without limitation, filtration,centrifugation, screening for hematopathologies, screening for viraland/or microbial infection, erythrocyte depletion, T-cell depletion toreduce incidence of graft-versus-host disease in allogenic stem celltransplant recipients, volume reduction, cell separation, resuspensionof cells in culture medium or a buffer suitable for subsequentprocessing, separation of stem cells from non-stem cells e.g., stem cellenrichment), ex vivo or in vitro stem cell expansion with growthfactors, cytokines, and/or hormones, and cryopreservation.

Any suitable method for stem cell enrichment known in the art may beused. Examples of stem cell enrichment methods include, withoutlimitation, fluorescence activated cell sorting (FACS) and magneticactivated cell sorting (MACS).

Accordingly, in certain embodiments, HSCs suitable for use in themethods of the present disclosure are human HSCs. In other embodiments,HSCs suitable for use in the methods of the present disclosure areautologous to the subject. In further embodiments, HSCs suitable for usein the methods of the present disclosure are allogenic to the subject.

HSCs obtained from a donor may be identified and/or enriched by anysuitable method of stem cell identification and enrichment known in theart, such as by utilizing certain phenotypic or genotypic markers. Forexample, in some embodiments, identification of HSCs includes using cellsurface markers associated with HSCs or specifically associated withterminally differentiated cells of the system. Suitable surface markersmay include, without limitation, one or more of c-kit, Sca-1, CD4, CD34,CD38, Thy1, CD2, CD3, CD4, CD5, CD8, CD43, CD45, CD59, CD90, CD105,CD133, CD135, ABCG2, NK1.1, B220, Ter-119, Flk-2, CDCP1, Endomucin,Gr-1, CD46, Mac-1, Thy1.1, and the signaling lymphocyte activationmolecule (SLAM) family of receptors. Examples of SLAM receptors include,without limitation, CD150, CD48, and CD244.

Additionally, HSCs obtained from a donor may be separated from non-stemcells by any suitable method known in the art including, withoutlimitation, fluorescence activated cell sorting (FACS) and magneticactivated cell sorting (MACS).

In one non-limiting example, human peripheral blood cells are incubatedwith antibodies recognizing c-kit, Sca-1, CD34, CD38, Thy1, CD2, CD3,CD4, CD5, CD8, CD43, CD45, CD59, CD90, CD105, CD133, ABCG2, NK1.1, B220,Ter-119, Flk-2, CDCP1, Endomucin, or Gr-1. Antibodies for CD2, CD3, CD4,CD5, CD8, NK1.1, B220, Ter-119, and Gr-1 are conjugated with magneticbeads. The cells expressing CD2, CD3, CD4, CD5, CD8, NK1.1, B220,Ter-119, or Gr-1 are retained in the column equipped to trap magneticbeads and cells attached to magnetic bead conjugated antibodies. Thecells that are not captured by the MACS column are subjected to FACSanalysis. Antibodies for c-kit, Sca-1, CD34, CD38, Thy1, are conjugatedwith fluorescent materials known in the art. The cells that are CD34+,CD38^(low/−), c-kit^(−/low), Thy1⁺ are separated from the rest of sampleby virtue of the types of fluorescent antibodies associated with thecells. These cells are provided as human long-term HSCs suitable for usewith any of the methods of the present disclosure.

In another non-limiting example, cells obtained from a subject arelabeled with the same set of magnetic bead conjugated antibodies asdescribed above (antibodies against one or more of CD2, CD3, CD4, CD5,CD8, NK1.1, B220, Ter-119, or Gr-1) and fluorescent conjugated CD150,CD244 and/or CD48 antibodies. After removing cells captured by themagnetic bead conjugated antibodies from the sample, the sample isanalyzed by FACS and CD150⁺, CD244⁻ and CD48⁻ cells are retained aslong-term HSCs.

In some embodiments, HSCs utilized in the methods of the presentdisclosure contain one or more of the markers: c-kit⁻, Sca-1⁺,CD34^(low/−), CD38⁺, Thy1^(+/low), CD34⁺, CD38^(low/−), c-kit^(−/low),and/or Thy1⁺. In some embodiments, the HSCs utilized in the methods ofthe present disclosure lack one or more of the markers: CD2, CD3, CD4,CD5, CD8, NK1.1, B220, Ter-119, and/or Gr-1. In certain embodiments, theHSCs utilized in the methods of the present disclosure are of an A⁺, A⁻,B⁺, B⁻, O⁺, O⁻, AB⁺, or AB⁻ type.

Alternatively, suitable HSCs may be obtained from a non-human source.Suitable non-human HSCs may be isolated from, femurs, hip, ribs,sternum, and other bones of a non-human animal, including, withoutlimitation, laboratory/research animals, rodents, pets, livestock, farmanimals, work animals, pack animals, rare or endangered species, racinganimals, and zoo animals. Further examples of suitable non-human animalsinclude, without limitation, monkeys, primates, mice, rats, guinea pigs,hamsters, dogs, cats, horses, cows, pigs, sheep, goats, and chickens.For example, HSCs may be obtained from murine bone marrow cells, byincubating the bone marrow cells with antibodies recognizing cellsurface molecules such as one or more of c-kit, Sca-1, CD34, CD38, Thy1,CD2, CD3, CD4, CD5, CD8, CD43, CD45, CD59, CD90, CD105, CD133, ABCG2,NK1.1, B220, Ter-119, Flk-2, CDCP1, Endomucin, or Gr-1. Antibodies forCD2, CD3, CD4, CD5, CDS, NK1.I, B220, Ter-119, and Gr-1 are conjugatedwith magnetic beads. In MACS equipment, the cells harboring CD2, CD3,CD4, CD5, CD8, NK1.1, B220, Ter-119, or Gr-1 on their surface areretained in the column equipped to trap magnetic beads and the cellsattached to magnetic bead conjugated antibodies. The cells that are notcaptured by MACS column are subjected to FACS analysis. For FACSanalysis, Antibodies for surface molecules such as c-kit, Sca-1, CD34,CD38, Thy1, are conjugated with fluorescent materials. The cells thatare c-kit⁺, Sca-1⁻, CD34^(low/−), CD38⁺, Thy1^(+/low) are separated fromthe rest of the sample by virtue of the types of fluorescent antibodiesassociated with the cells. These cells are provided as murine long-termHSCs suitable for use with any of the methods of the present disclosure.In other embodiments, different sets of marker are used to separatemurine long-term HSCs from cells of bone marrow, umbilical cord blood,fetal tissue, and peripheral blood.

In some embodiments, obtaining HSCs from bone marrow includes firstinjecting the HSC donor, such as a mouse, with 5-fluorouracil (5-FU) toinduce the HSCs to proliferate in order to enrich for HSCs in the bonemarrow of the donor.

Moreover, HSCs suitable for use with any of the methods of the presentdisclosure, whether obtained from, or present in cord blood, bonemarrow, peripheral blood, or other source, may be grown or expanded inany suitable, commercially available or custom defined medium, with orwithout serum, as desired (e.g., Hartshorn et al., Cell Technology forCell Products, pages 221-224, R. Smith, Editor; Springer Netherlands,2007). For example, serum free medium may utilize albumin and/ortransferrin, which have been shown to be useful for the growth andexpansion of CD34⁺ cells in serum free medium. Also, cytokines may beincluded, such as Flt-3 ligand, stem cell factor (SCF), andthrombopoietin (TPO), among others. HSCs may also be grown in vesselssuch as bioreactors (e.g., Liu et al., Journal of Biotechnology124:592-601, 2006). A suitable medium for ex vivo expansion of HSCs mayalso contain HSC supporting cells, such as stromal cells (e.g.,lymphoreticular stromal cells), which can be derived, for example, fromthe disaggregation of lymphoid tissue, and which have been shown tosupport the in vitro, ex vivo, and in vivo maintenance, growth, anddifferentiation of HSCs, as well as their progeny.

HSC growth or expansion may be measured in vitro or in vivo according toroutine techniques known in the art. For example, WO 2008/073748,describes methods for measuring in vivo and in vitro expansion of HSCs,and for distinguishing between the growth/expansion of HSCs and thegrowth/expansion of other cells in a potentially heterogeneouspopulation (e.g., bone marrow), such as intermediate progenitor cells.

HSC Cell Lines

In other embodiments, HSCs suitable for use in any of the methods of thepresent disclosure may also be derived from an HSC cell line. SuitableHSC cell lines include any cultured hematopoietic stem cell line knownin the art. Non-limiting examples include the conditionally immortalizedlong-term stem cell lines described in U.S. Patent ApplicationPublication Nos. US 2007/0116691 and US 2010/0047217.

In certain embodiments, HSCs suitable for use in the methods of thepresent disclosure are conditionally immortalized before administeringthe HSCs to a subject. For example, HSCs obtained by any methoddisclosed herein may be treated with a regulatable (e.g., inducible,controllable) protooncogene that promotes cell survival andproliferation, such as MYC, and/or with a protein that inhibitsapoptosis of the HSCs, such as Bcl-2 (e.g., U.S. Patent ApplicationPublication No. US 2007/0116691). In some embodiments, the regulatableprotooncogene is a MYC-composition. Preferably, the MYC-composition is aTAT-MYC fusion protein. The protein that inhibits apoptosis of the HSCsmay also be regulatable. For example, the regulatable protein may be aPTD-Bcl-2 fusion protein, such as a TAT-Bcl-2 fusion protein.

In other embodiments, HSCs suitable for use in the methods of thepresent disclosure are cultured in the presence of a MYC-composition(e.g., a TAT-MYC protein fusion) of the present disclosure, aBcl-2-composition (e.g., a TAT-BC1-2 protein fusion) of the presentdisclosure, or both before administering the HSCs to a subject.Preferably, culturing the first composition in the presence of theMYC-composition and/or Bcl-2-composition conditionally immortalizes theHSCs, which results in an expansion of the cultured HSCs.

As used herein, an “expansion of hematopoietic stem cells” or an“expansion of HSCs” refers to an increase in cell proliferation and/orcell survival, as compared to HSCs that have not been cultured ortreated with a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both. For example, HSCexpansion occurs when HSC proliferation, HSC survival, or both isincreased by at least about or about 10% to at least about or about500%. Any method known in the art and disclosed herein for measuring anincrease in HSC proliferation and/or survival may be used.

Accordingly, HSCs suitable for use in any of the methods of the presentdisclosure may be obtained from bone marrow, from an apheresisprocedure, from peripheral blood cells, from peripheral blood cells thathave undergone leukapheresis, from umbilical cord blood, from amnioticfluid, from cultured HSC cells, from an immortalized HSC cell line, orfrom a conditionally immortalized HSC cell line. Alternatively, HSCssuitable for use in any of the methods of the present disclosure may bepresent in bone marrow, in peripheral blood cells, in peripheral bloodcells that have undergone leukapheresis, in umbilical cord blood, inamniotic fluid, and in cell lines.

Transgenic Approach

In some embodiments, conditionally immortalized HSCs for use in themethods of the present disclosure are established using any transgenicapproach known in the art (e.g., U.S. Patent Application PublicationNos. US 2007/0116691 and US 2010/0047217. For example, HSCs may beimmortalized by obtaining an expanded population of HSCs, transfecting(transducing) the HSCs with a vector that encodes, for example, a MYCpolypeptide and/or a Bcl-2 polypeptide (e.g., inducible and/orcontrollable), transfecting (transducing) the HSCs with a vectorencoding the MYC polypeptide and/or the Bcl-2 polypeptide, and expandingthe transfected HSCs in the presence of a combination of stem cellgrowth factors under conditions where the MYC polypeptide and/or Bcl-2polypeptide is induced and/or active.

The MYC polypeptide and/or Bcl-2 polypeptide is regulatable (e.g.,inducible or controllable), so that the polypeptide can be activated anddeactivated (i.e., turned on or turned off) as desired to eithermaintain the HSCs in an immortalized state or to allow it todifferentiate into a desired cell type.

In some embodiments, the MYC polypeptide and/or Bcl-2 polypeptide hasbeen modified such that activity is inducible or repressible. Forexample, the MYC polypeptide and/or Bcl-2 polypeptide may furthercontain an inducible receptor. In certain embodiments, the recombinantproteins contain an estrogen receptor (ER). In certain embodiments, therecombinant protein that promotes cell survival and/or proliferation andthat contains an estrogen receptor is a MYC-ER polypeptide and/or aBcl-2-ER polypeptide. In certain embodiments, the proteins containing anestrogen receptor are induced by 4-hydroxytamoxifen (4-OHT).Alternatively, the proteins may contain a glucocorticoid receptor (GR),e.g., a glucocorticoid receptor that is sensitive to mifepristone(MIFEPREX). In certain embodiments, the protein that contains aglucocorticoid receptor is a MYC-GR polypeptide and/or a Bcl-2-GRpolypeptide.

Any method known in the art for obtaining an expanded population of HSCsknown in the art may be used. For example, HSCs may be cultured with oneor more growth factor that promotes cell proliferation and/or celldivision.

Preferably, the vectors are an integrating vector, which has the abilityto integrate into the genome of a cell (e.g., a retroviral vector). TheHSCs can be transfected and/or transduce with the vectors using anysuitable method of transfecting cells, and particularly mammalian cells,including by using combinations of techniques. Examples of suitablevectors, include without limitation, retroviral vectors, lentivirusvectors, parvovirus vectors, vaccinia virus vectors, coronavirusvectors, calicivirus vectors, papilloma virus vectors, flavivirusvectors, orthomixovirus vectors, togavirus vectors, picornavirusvectors, adenoviral vectors, and modified and attenuated herpesvirusesvectors. Any such virus vector can further be modified with specificsurface expressed molecules that target these to HSCs, such as membranebound SCF, or other stem-cell specific growth factor ligands. Othermethods of transfection of mammalian cells include, but are not limitedto, direct electroporation of mammalian expression vectors, such as byusing NUCLEOFECTOR™ technology (AMAXA Biosystems). This technology is ahighly efficient non-viral gene transfer method for most primary cellsand for hard-to-transfect cell lines, which is an improvement on thelong-known method of electroporation, based on the use of cell-typespecific combinations of electrical current and solutions to transferpolyanionic macromolecules directly into the nucleus. Additionally,suitable methods of transfection can include any bacterial, yeast, orother artificial methods of gene delivery that are known in the art.

Enhancement of Endogenous Expression

In some embodiments, conditionally immortalized HSCs for use in themethods of the present disclosure may be established by enhancing theexpression of endogenous proteins that promote cell survival and/orproliferation, including, without limitation, any MYC protein of thepresent disclosure. Additionally, conditionally immortalized HSCs foruse in the methods of the present disclosure may be established by alsoenhancing the expression of endogenous proteins that inhibit apoptosis,including, without limitation, any Bcl-2 protein of the presentdisclosure.

Protein Transduction Approach

In some embodiments, HSCs obtained and/or produced by any methoddisclosed herein may be treated with a gene product that promotes cellsurvival and/or proliferation, including, but not limited to any MYCprotein of the present disclosure, and/or with a protein that inhibitsapoptosis of the HSCs, including, but limited to a Bcl-2 protein of thepresent disclosure. In some embodiments, the MYC protein is a fusionprotein containing a PTD. In some embodiments, the Bcl-2 protein is afusion protein containing a PTD. In some embodiments, HSCs obtainedand/or produced by any method disclosed herein may be treated with oneor more compound (optionally an exogenous protein) that enables thetransient upregulation of at least one function of a MYC protein of thepresent disclosure. In some embodiments, the MYC protein is a PTD-MYCfusion protein. In certain embodiments, the PTD-MYC fusion protein is aTAT-MYC fusion protein.

In some embodiments, HSCs obtained by any method disclosed herein may betreated with one or more compound (optionally an exogenous protein) thatenables the transient upregulation of at least one function of a Bcl-2protein of the present disclosure. In some embodiments, the exogenousBcl-2 protein is a PTD-Bcl-2 fusion protein. In some embodiments, thePTD-Bcl-2 fusion protein is a TAT-Bcl-2 fusion protein.

In other embodiments, HSCs suitable for use in any of the methods of thepresent disclosure are treated with a composition containing a fusionprotein containing a MYC protein of the present disclosure fused to aPTD (e.g., a PTD-MYC fusion protein), a composition containing a fusionprotein containing a Bcl-2 protein of the present disclosure fused to aPTD (e.g., a PTD-Bcl-2 fusion protein), or both.

Accordingly, HSCs suitable for use in any of the methods of the presentdisclosure may be obtained from embryonic stem cells (ES cells), fetalstem cells, induced pluripotent stem cells (iPS cells), bone marrow,from peripheral blood cells, from peripheral blood cells that haveundergone apheresis, from peripheral blood cells that have undergoneleukapheresis, from umbilical cord blood, from amniotic fluid, fromcultured HSC cells, from an immortalized HSC cell line, or from aconditionally immortalized HSC cell line.

HSC Compositions

In other embodiments, HSCs suitable for use in any of the methods of thepresent disclosure are included in a composition. SuitableHSC-containing compositions may contain, without limitation, isolatedand/or purified HSCs; whole bone marrow; bone marrow enriched for HSCs(e.g., 5-FU treated bone marrow); peripheral blood; and umbilical cordblood. In some embodiments, such compositions include a pharmaceuticallyacceptable carrier. Suitable pharmaceutically acceptable carriers mayinclude pharmaceutically acceptable excipients and/or delivery vehicles,for delivering the HSCs to a subject, such as a patient. Additionally,pharmaceutically acceptable carriers may contain a cell culture mediumthat supports HSC viability. The medium will generally be serum-free inorder to avoid provoking an immune response in the recipient. Thecarrier will generally be buffered and/or pyrogen-free.

In some embodiments, a therapeutically effective amount of a compositioncontaining HSCs is administered to a subject to achieve hematopoieticcompartment reconstitution in the subject. Generally, administering 10⁴to 10⁶ HSCs is sufficient to achieve hematopoietic compartmentreconstitution. In certain embodiments, administering a MYC-compositionof the present disclosure, a Bcl-2-composition of the presentdisclosure, or both to an HSC transplant recipient can reduce the numberof HSCs required to achieve hematopoietic compartment reconstitution.For example, administering a therapeutically effective amount of aMYC-composition of the present disclosure, a Bcl-2-composition of thepresent disclosure, or both to an HSC transplant recipient can reducethe therapeutically effective amount of HSCs in an HSC composition ofthe present disclosure required to achieve hematopoietic compartmentreconstitution by at least about or about 10% to least about or about75%, compared to the amount of HSCs required to achieve hematopoieticcompartment reconstitution in an HSC transplant recipient that is notadministered the MYC-composition and/or Bcl-2-composition.

In certain embodiments, administering a therapeutically effective amountof a MYC-composition of the present disclosure, a Bcl-2-composition ofthe present disclosure, or both to an HSC transplant recipient canreduce the therapeutically effective amount of HSCs in an HSCcomposition of the present disclosure required to achieve hematopoieticcompartment reconstitution by at least about or about 10% to at leastabout or about 75%, for example, by at least about or about 10%, atleast about or about 15%, at least about or about 20%, at least about orabout 25%, at least about or about 30%, at least about or about 35%, atleast about or about 40%, at least about or about 50%, at least about orabout 55%, at least about or about 60%, at least about or about 65%, atleast about or about 70%, at least about or about 75%, or a higherpercentage less, as compared to the amount of HSCs required to achievehematopoietic compartment reconstitution in an HSC transplant recipientthat is not administered the MYC-composition and/or Bcl-2-composition.

In certain embodiments, treating a population with a therapeuticallyeffective amount of a MYC-composition, a Bcl-2-composition, or both to apopulation of HSCs prior to the HSCs being transplanted to subject inneed thereof can reduce the therapeutically effective amount of HSCsrequired to achieve hematopoietic compartment reconstitution by at leastabout or about 10% to at least about or about 75%, for example, by atleast about or about 10%, at least about or about 15%, at least about orabout 20%, at least about or about 25%, at least about or about 30%, atleast about or about 35%, at least about or about 40%, at least about orabout 50%, at least about or about 55%, at least about or about 60%, atleast about or about 65%, at least about or about 70%, at least about orabout 75%, or a higher percentage less, as compared to the amount ofHSCs required to achieve hematopoietic compartment reconstitution in anHSC transplant recipient that does receive HSCs that were pre-treatedwith a MYC-composition of the present disclosure, a Bcl-2-composition ofthe present disclosure, or both.

HSC compositions and populations of HSCs of the present disclosure aregenerally administered into the body of a subject, such as a patient, byany suitable methods known in the art, including without limitation,injection and implantation. For example, HSC may be directly injectedinto the tissue in which they are intended to act using a syringecontaining an HSC composition of the present disclosure. Alternatively,an HSC composition of the present disclosure may be delivered via acatheter, such as a central venous catheter, attached to a syringecontaining the HSC composition.

HSC Transplantation

In further embodiments, HSC-containing compositions and populations ofHSCs of the present disclosure are administered to a subject in need ofhematopoietic stem cell (HSC) transplantation as a step in the processof an HSC transplantation procedure. In certain preferred embodiments,the subject is a human patient in need of a HSC transplant. In otherembodiments, the subject is any non-human animal, including, withoutlimitation, laboratory/research animals, rodents, pets, livestock, farmanimals, work animals, pack animals, rare or endangered species, racinganimals, zoo animals, monkeys, primates, mice, rats, guinea pigs,hamsters, dogs, cats, horses, cows, pigs, sheep, goats, and chickens.

The HSC transplantation procedure may be a myeloablative HSC transplant.Myeloablation generally refers to the ablation or suppression of theendogenous hematopoietic compartment of an HSC transplant recipient.Myeloablation occurs prior to HSC transplantation. In HSC transplantrecipients suffering from a hematological disease, such as ahematological cancer, myeloablation may be performed to help eradicatethe disease. Myeloablation may also be performed to suppress theendogenous immune system of the HSC transplant recipient in order tohelp reduce the risk of rejection of the transplanted HSCs (e.g.,graft-versus-host disease). Any method known in the art formyeloablation may be used. Examples of myeloablation procedures include,without limitation, chemotherapy, irradiation, and combinations thereof.

Alternatively, the HSC transplantation procedure may benon-myeloablative. In non-myeloablative procedures lower doses ofchemotherapy and/or radiation are used in the recipient prior to HSCtransplantation.

Subjects in need of an HSC transplant include subjects presenting withan HSC transplant indication. Examples of HSC transplant indicationsinclude, without limitation, a hematological malignancy, a myeloma,multiple myeloma, a leukemia, acute lymphoblastic leukemia, chroniclymphocytic leukemia, a lymphoma, indolent lymphoma, non-Hodgkinlymphoma, diffuse B cell lymphoma, follicular lymphoma, mantle celllymphoma, T cell lymphoma, Hodgkin lymphoma, a neuroblastoma, aretinoblastoma, Shwachman Diamond syndrome, a brain tumor, Ewing'sSarcoma, a Desmoplastic small round cell tumor, a relapsed germ celltumor, a hematological disorder, a hemoglobinopathy, an autoimmunedisorder, juvenile idiopathic arthritis, systemic lupus erythematosus,severe combined immunodeficiency, congenital neutropenia with defectivestem cells, severe aplastic anemia, a sickle-cell disease, amyelodysplastic syndrome, chronic granulomatous disease, a metabolicdisorder, Hurler syndrome, Gaucher disease, osteopetrosis, malignantinfantile osteopetrosis, heart disease, HIV, and AIDS. Additionally, asubject in need of an HSC transplant can also include a subject that hashad an organ transplant.

Enhancing Hematopoietic Compartment Cell Formation

Other aspects of the present disclosure relate to enhancinghematopoietic compartment cell formation in a subject in need thereof byadministering a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both; and/oradministering a population of HSCs that has been pre-treated with aMYC-composition of the present disclosure, a Bcl-2-composition of thepresent disclosure, or both. In some embodiments, enhancinghematopoietic compartment cell formation includes enhancinghematopoietic compartment reconstitution. In some embodiments, enhancinghematopoietic compartment cell formation includes enhancinghematopoietic compartment autoreconstitution.

Enhancing Hematopoietic Compartment Reconstitution

In certain embodiments, administering a population of HSCs that havebeen pre-treated with a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both, to a subject inneed of HSC transplantation enhances hematopoietic compartmentreconstitution in the subject. In other embodiments, administering aMYC-composition of the present disclosure, a Bcl-2-composition of thepresent disclosure, or both to a subject that has received or willreceive an HSC transplant enhances hematopoietic compartmentreconstitution in the subject. For example, pre-treating HSCs with theMYC-composition, the Bcl-2-composition, or both, and then administeringthe HSCs; or administering the MYC-composition, the Bcl-2-composition,or both may improve the rate of hematopoietic compartmentreconstitution, increase the amount of hematopoietic compartment cellsthat are formed, or reduce loss of hematopoietic compartment cells inthe subject, as compared to a subject that is administered HSCs thatwere not pre-treated with the MYC-composition, the Bcl-2-composition, orboth; or was not administered the MYC-composition, theBcl-2-composition, or both.

Preferably, pre-treating the HSCs with the MYC-composition, theBcl-2-composition, or both prior to administering the HSCs to a subject;or administering the MYC-composition, the Bcl-2-composition, or both tothe subject accelerates (i.e., increases the rate of) hematopoieticcompartment reconstitution in the subject, as compared to hematopoieticcompartment reconstitution in a subject that has not been administeredthe pre-treated HSCs or the MYC-composition, the Bcl-2-composition, orboth.

As used herein, an “acceleration in hematopoietic compartmentreconstitution” refers to a reduction in the time required toreconstitute at least about or about 25% to about 100% of one or more ofthe blood cell lineages in the hematopoietic compartment, including,without limitation, monocytes, macrophages, neutrophils, basophils,eosinophils, erythrocytes, megakaryocytes, platelets, and dendriticcells; and the lymphoid lineage, which includes, without limitation,T-cells, B-cells, NKT-cells, and NK cells. For example, a reduction from8 weeks to 4 weeks in the time required to reconstitute thehematopoietic compartment would constitute an acceleration of 50%.

Pre-treatment with a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both; or administrationof a MYC-composition of the present disclosure, a Bcl-2-composition ofthe present disclosure, or both may achieve an at least about or about25% to art least about or about 500%, for example, an at least about orabout 25%, at least about or about 30%, at least about or about 31%, atleast about or about or about 32%, at least about or about 33%, at leastabout or about 34%, at least about or about 35%, at least about or about40%, at least about or about 45%, at least about or about 50%, at leastabout or about 55%, at least about or about 60%, at least about or about65%, at least about or about 66%, at least about or about 67%, at leastabout or about 68%, at least about or about 69%, at least about or about70%, at least about or about 75%, at least about or about 80%, at leastabout or about 90%, at least about or about 95%, at least about or about100%, at least about or about 150%, at least about or about 200%, atleast about or about 250%, at least about or about 300%, at least aboutor about 400%, at least about or about 500%, or a higher percentageacceleration in hematopoietic compartment reconstitution in a subjectthat has received or will receive an HSC transplant, compared tohematopoietic compartment reconstitution in a subject that is notadministered the pre-treated HSCs; or the MYC-composition, theBcl-2-composition, or both.

As disclosed herein, hematopoietic compartment reconstitution includes,without limitation, T-cell compartment reconstitution, B-cellcompartment reconstitution, NK-cell compartment reconstitution, myeloidcell compartment reconstitution, and neutrophil recovery. As usedherein, the term “T-cell compartment” refers to the cell compartment ina subject that contains all immature, mature, undifferentiated anddifferentiated B-cells. The “T-cell compartment” includes, withoutlimitation, T-cell progenitors, pro-T-cells, pre-T-cells, doublepositive T-cells, immature T-cells, mature T-cells, helper T-cells,cytotoxic T-cells, memory T-cells, regulatory T-cells, natural killerT-cells (NKT-cells), and gamma delta T-cells. As used herein, the term“B-cell compartment” refers to the cell compartment in a subject thatcontains all immature, mature, undifferentiated and differentiatedB-cells. The “B-cell compartment” includes, without limitation, B-cellprogenitors, pro-B-cells, early pro-B-cells, late-pro B-cells, largepre-B-cells, small pre-B-cells, immature B-cells, mature B-cells, plasmaB-cells, memory B-cells, B-1 cells, B-2 cells, marginal-zone B-cells,and follicular B-cells. As used herein, the term “NK cell compartment”refers to the cell compartment in a subject that contains allprogenitor, immature, mature, undifferentiated and differentiatednatural killer (NK) cells. The “NK cell compartment” NK cells. As usedherein, the term “myeloid cell compartment” refers to the cellcompartment in a subject that contains all progenitor, immature, mature,undifferentiated and differentiated myeloid cell populations. The“myeloid cell compartment” includes, without limitation, monocytes,macrophages, neutrophils, basophils, eosinophils, erythrocytes,megakaryocytes, platelets, and dendritic cells.

Accordingly, in certain embodiments, the accelerated hematopoieticcompartment reconstitution in an HSC transplant recipient that has beenadministered HSCs pre-treated with a MYC-composition of the presentdisclosure, a Bcl-2-composition of the present disclosure, or both; oradministered a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both results in T-cellcompartment reconstitution that is accelerated by at least about orabout 25% to art least about or about 500%, for example, at least aboutor about 25%, at least about or about 30%, at least about or about 31%,at least about or about 32%, at least about or about 33%, at least aboutor about 34%, at least about or about 35%, at least about or about 40%,at least about or about 45%, at least about or about 50%, at least aboutor about 55%, at least about or about 60%, at least about or about 65%,at least about or about 66%, at least about or about 67%, at least aboutor about 68%, at least about or about 69%, at least about or about 70%,at least about or about 75%, at least about or about 80%, at least aboutor about 90%, at least about or about 95%, at least about or about 100%,at least about or about 150%, at least about or about 200%, at leastabout or about 250%, at least about or about 300%, at least about orabout 400%, at least about or about 500%, or a higher percentage more,compared to T-cell compartment reconstitution in a subject that is notadministered the pre-treated HSCs; or the MYC-composition, theBcl-2-composition, or both.

In other embodiments, the accelerated hematopoietic compartmentreconstitution in an HSC transplant recipient that has been administeredHSCs pre-treated with a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both; or administered aMYC-composition of the present disclosure, a Bcl-2-composition of thepresent disclosure, or both results in NKT-cell reconstitution that isaccelerated by at least about or about 25% to art least about or about500%, for example, at least about or about 25%, at least about or about30%, at least about or about 31%, at least about or about 32%, at leastabout or about 33%, at least about or about 34%, at least about or about35%, at least about or about 40%, at least about or about 45%, at leastabout or about 50%, at least about or about 55%, at least about or about60%, at least about or about 65%, at least about or about 66%, at leastabout or about 67%, at least about or about 68%, at least about or about69%, at least about or about 70%, at least about or about 75%, at leastabout or about 80%, at least about or about 90%, at least about or about95%, at least about or about 100%, at least about or about 150%, atleast about or about 200%, at least about or about 250%, at least aboutor about 300%, at least about or about 400%, at least about or about500%, or a higher percentage more, compared to NKT-cell reconstitutionin a subject that is not administered the pre-treated HSCs; or theMYC-composition, the Bcl-2-composition, or both.

In other embodiments, the accelerated hematopoietic compartmentreconstitution in an HSC transplant recipient that has been administeredHSCs pre-treated with a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both; or administered aMYC-composition of the present disclosure, a Bcl-2-composition of thepresent disclosure, or both results in B-cell compartment reconstitutionthat is accelerated by at least about or about 25% to art least about orabout 500%, for example, at least about or about 25%, at least about orabout 30%, at least about or about 31%, at least about or about 32%, atleast about or about 33%, at least about or about 34%, at least about orabout 35%, at least about or about 40%, at least about or about 45%, atleast about or about 50%, at least about or about 55%, at least about orabout 60%, at least about or about 65%, at least about or about 66%, atleast about or about 67%, at least about or about 68%, at least about orabout 69%, at least about or about 70%, at least about or about 75%, atleast about or about 80%, at least about or about 90%, at least about orabout 95%, at least about or about 100%, at least about or about 150%,at least about or about 200%, at least about or about 250%, at leastabout or about 300%, at least about or about 400%, at least about orabout 500%, or a higher percentage more, compared to B-cell compartmentreconstitution in a subject that is not administered the pre-treatedHSCs; or the MYC-composition, the Bcl-2-composition, or both.

In other embodiments, the accelerated hematopoietic compartmentreconstitution in an HSC transplant recipient that has been administeredHSCs pre-treated with a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both; or administered aMYC-composition of the present disclosure, a Bcl-2-composition of thepresent disclosure, or both results in NK-cell compartmentreconstitution that is accelerated by at least about or about 25% to artleast about or about 500%, for example, at least about or about 25%, atleast about or about 30%, at least about or about 31%, at least about orabout 32%, at least about or about 33%, at least about or about 34%, atleast about or about 35%, at least about or about 40%, at least about orabout 45%, at least about or about 50%, at least about or about 55%, atleast about or about 60%, at least about or about 65%, at least about orabout 66%, at least about or about 67%, at least about or about 68%, atleast about or about 69%, at least about or about 70%, at least about orabout 75%, at least about or about 80%, at least about or about 90%, atleast about or about 95%, at least about or about 100%, at least aboutor about 150%, at least about or about 200%, at least about or about250%, at least about or about 300%, at least about or about 400%, atleast about or about 500%, or a higher percentage more, compared toNK-cell compartment reconstitution in a subject that is not administeredthe pre-treated HSCs; or the MYC-composition, the Bcl-2-composition, orboth.

In other embodiments, the accelerated hematopoietic compartmentreconstitution in an HSC transplant recipient that has been administeredHSCs pre-treated with a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both; or administered aMYC-composition of the present disclosure, a Bcl-2-composition of thepresent disclosure, or both results in myeloid cell compartmentreconstitution that is accelerated by at least about or about 25% to artleast about or about 500%, for example, at least about or about 25%, atleast about or about 30%, at least about or about 31%, at least about orabout 32%, at least about or about 33%, at least about or about 34%, atleast about or about 35%, at least about or about 40%, at least about orabout 45%, at least about or about 50%, at least about or about 55%, atleast about or about 60%, at least about or about 65%, at least about orabout 66%, at least about or about 67%, at least about or about 68%, atleast about or about 69%, at least about or about 70%, at least about orabout 75%, at least about or about 80%, at least about or about 90%, atleast about or about 95%, at least about or about 100%, at least aboutor about 150%, at least about or about 200%, at least about or about250%, at least about or about 300%, at least about or about 400%, atleast about or about 500%, or a higher percentage more, compared tomyeloid cell compartment reconstitution in a subject that is notadministered the pre-treated HSCs; or the MYC-composition, theBcl-2-composition, or both.

In other embodiments, the accelerated hematopoietic compartmentreconstitution in an HSC transplant recipient that has been administeredHSCs pre-treated with a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both; or administered aMYC-composition of the present disclosure, a Bcl-2-composition of thepresent disclosure, or both results in monocyte reconstitution that isaccelerated by at least about or about 25% to art least about or about500%, for example, at least about or about 25%, at least about or about30%, at least about or about 31%, at least about or about 32%, at leastabout or about 33%, at least about or about 34%, at least about or about35%, at least about or about 40%, at least about or about 45%, at leastabout or about 50%, at least about or about 55%, at least about or about60%, at least about or about 65%, at least about or about 66%, at leastabout or about 67%, at least about or about 68%, at least about or about69%, at least about or about 70%, at least about or about 75%, at leastabout or about 80%, at least about or about 90%, at least about or about95%, at least about or about 100%, at least about or about 150%, atleast about or about 200%, at least about or about 250%, at least aboutor about 300%, at least about or about 400%, at least about or about500%, or a higher percentage more, compared to monocyte reconstitutionin a subject that is not administered the pre-treated HSCs; or theMYC-composition, the Bcl-2-composition, or both.

In other embodiments, the accelerated hematopoietic compartmentreconstitution in an HSC transplant recipient that has been administeredHSCs pre-treated with a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both; or administered aMYC-composition of the present disclosure, a Bcl-2-composition of thepresent disclosure, or both results in macrophage reconstitution that isaccelerated by at least about or about 25% to art least about or about500%, for example, at least about or about 25%, at least about or about30%, at least about or about 31%, at least about or about 32%, at leastabout or about 33%, at least about or about 34%, at least about or about35%, at least about or about 40%, at least about or about 45%, at leastabout or about 50%, at least about or about 55%, at least about or about60%, at least about or about 65%, at least about or about 66%, at leastabout or about 67%, at least about or about 68%, at least about or about69%, at least about or about 70%, at least about or about 75%, at leastabout or about 80%, at least about or about 90%, at least about or about95%, at least about or about 100%, at least about or about 150%, atleast about or about 200%, at least about or about 250%, at least aboutor about 300%, at least about or about 400%, at least about or about500%, or a higher percentage more, compared to macrophage reconstitutionin a subject that is not administered the pre-treated HSCs; or theMYC-composition, the Bcl-2-composition, or both.

In other embodiments, the accelerated hematopoietic compartmentreconstitution in an HSC transplant recipient that has been administeredHSCs pre-treated with a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both; or administered aMYC-composition of the present disclosure, a Bcl-2-composition of thepresent disclosure, or both results in basophil reconstitution that isaccelerated by at least about or about 25% to art least about or about500%, for example, at least about or about 25%, at least about or about30%, at least about or about 31%, at least about or about 32%, at leastabout or about 33%, at least about or about 34%, at least about or about35%, at least about or about 40%, at least about or about 45%, at leastabout or about 50%, at least about or about 55%, at least about or about60%, at least about or about 65%, at least about or about 66%, at leastabout or about 67%, at least about or about 68%, at least about or about69%, at least about or about 70%, at least about or about 75%, at leastabout or about 80%, at least about or about 90%, at least about or about95%, at least about or about 100%, at least about or about 150%, atleast about or about 200%, at least about or about 250%, at least aboutor about 300%, at least about or about 400%, at least about or about500%, or a higher percentage more, compared to basophil reconstitutionin a subject that is not administered the pre-treated HSCs; or theMYC-composition, the Bcl-2-composition, or both.

In other embodiments, the accelerated hematopoietic compartmentreconstitution in an HSC transplant recipient that has been administeredHSCs pre-treated with a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both; or administered aMYC-composition of the present disclosure, a Bcl-2-composition of thepresent disclosure, or both results in eosinophil reconstitution that isaccelerated by at least about or about 25% to art least about or about500%, for example, at least about or about 25%, at least about or about30%, at least about or about 31%, at least about or about 32%, at leastabout or about 33%, at least about or about 34%, at least about or about35%, at least about or about 40%, at least about or about 45%, at leastabout or about 50%, at least about or about 55%, at least about or about60%, at least about or about 65%, at least about or about 66%, at leastabout or about 67%, at least about or about 68%, at least about or about69%, at least about or about 70%, at least about or about 75%, at leastabout or about 80%, at least about or about 90%, at least about or about95%, at least about or about 100%, at least about or about 150%, atleast about or about 200%, at least about or about 250%, at least aboutor about 300%, at least about or about 400%, at least about or about500%, or a higher percentage more, compared to eosinophil reconstitutionin a subject that is not administered the pre-treated HSCs; or theMYC-composition, the Bcl-2-composition, or both.

In other embodiments, the accelerated hematopoietic compartmentreconstitution in an HSC transplant recipient that has been administeredHSCs pre-treated with a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both; or administered aMYC-composition of the present disclosure, a Bcl-2-composition of thepresent disclosure, or both results in erythrocyte reconstitution thatis accelerated by at least about or about 25% to art least about orabout 500%, for example, at least about or about 25%, at least about orabout 30%, at least about or about 31%, at least about or about 32%, atleast about or about 33%, at least about or about 34%, at least about orabout 35%, at least about or about 40%, at least about or about 45%, atleast about or about 50%, at least about or about 55%, at least about orabout 60%, at least about or about 65%, at least about or about 66%, atleast about or about 67%, at least about or about 68%, at least about orabout 69%, at least about or about 70%, at least about or about 75%, atleast about or about 80%, at least about or about 90%, at least about orabout 95%, at least about or about 100%, at least about or about 150%,at least about or about 200%, at least about or about 250%, at leastabout or about 300%, at least about or about 400%, at least about orabout 500%, or a higher percentage more, compared to erythrocytereconstitution in a subject that is not administered the pre-treatedHSCs; or the MYC-composition, the Bcl-2-composition, or both.

In other embodiments, the accelerated hematopoietic compartmentreconstitution in an HSC transplant recipient that has been administeredHSCs pre-treated with a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both; or administered aMYC-composition of the present disclosure, a Bcl-2-composition of thepresent disclosure, or both results in megakaryocyte reconstitution thatis accelerated by at least about or about 25% to art least about orabout 500%, for example, at least about or about 25%, at least about orabout 30%, at least about or about 31%, at least about or about 32%, atleast about or about 33%, at least about or about 34%, at least about orabout 35%, at least about or about 40%, at least about or about 45%, atleast about or about 50%, at least about or about 55%, at least about orabout 60%, at least about or about 65%, at least about or about 66%, atleast about or about 67%, at least about or about 68%, at least about orabout 69%, at least about or about 70%, at least about or about 75%, atleast about or about 80%, at least about or about 90%, at least about orabout 95%, at least about or about 100%, at least about or about 150%,at least about or about 200%, at least about or about 250%, at leastabout or about 300%, at least about or about 400%, at least about orabout 500%, or a higher percentage more, compared to megakaryocytereconstitution in a subject that is not administered the pre-treatedHSCs; or the MYC-composition, the Bcl-2-composition, or both.

In other embodiments, the accelerated hematopoietic compartmentreconstitution in an HSC transplant recipient that has been administeredHSCs pre-treated with a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both; or administered aMYC-composition of the present disclosure, a Bcl-2-composition of thepresent disclosure, or both results in platelet reconstitution that isaccelerated by at least about or about 25% to art least about or about500%, for example, at least about or about 25%, at least about or about30%, at least about or about 31%, at least about or about 32%, at leastabout or about 33%, at least about or about 34%, at least about or about35%, at least about or about 40%, at least about or about 45%, at leastabout or about 50%, at least about or about 55%, at least about or about60%, at least about or about 65%, at least about or about 66%, at leastabout or about 67%, at least about or about 68%, at least about or about69%, at least about or about 70%, at least about or about 75%, at leastabout or about 80%, at least about or about 90%, at least about or about95%, at least about or about 100%, at least about or about 150%, atleast about or about 200%, at least about or about 250%, at least aboutor about 300%, at least about or about 400%, at least about or about500%, or a higher percentage more, compared to platelet reconstitutionin a subject that is not administered the pre-treated HSCs; or theMYC-composition, the Bcl-2-composition, or both.

In other embodiments, the accelerated hematopoietic compartmentreconstitution in an HSC transplant recipient that has been administeredHSCs pre-treated with a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both; or administered aMYC-composition of the present disclosure, a Bcl-2-composition of thepresent disclosure, or both results in dendritic cell reconstitutionthat is accelerated by at least about or about 25% to art least about orabout 500%, for example, at least about or about 25%, at least about orabout 30%, at least about or about 31%, at least about or about 32%, atleast about or about 33%, at least about or about 34%, at least about orabout 35%, at least about or about 40%, at least about or about 45%, atleast about or about 50%, at least about or about 55%, at least about orabout 60%, at least about or about 65%, at least about or about 66%, atleast about or about 67%, at least about or about 68%, at least about orabout 69%, at least about or about 70%, at least about or about 75%, atleast about or about 80%, at least about or about 90%, at least about orabout 95%, at least about or about 100%, at least about or about 150%,at least about or about 200%, at least about or about 250%, at leastabout or about 300%, at least about or about 400%, at least about orabout 500%, or a higher percentage more, compared to dendritic cellreconstitution in a subject that is not administered the pre-treatedHSCs; or the MYC-composition, the Bcl-2-composition, or both.

In other embodiments, the accelerated hematopoietic compartmentreconstitution in an HSC transplant recipient that has been administeredHSCs pre-treated with a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both; or administered aMYC-composition of the present disclosure, a Bcl-2-composition of thepresent disclosure, or both results in neutrophil recovery that isaccelerated by at least about or about 25% to art least about or about500%, for example, at least about or about 25%, at least about or about30%, at least about or about 31%, at least about or about 32%, at leastabout or about 33%, at least about or about 34%, at least about or about35%, at least about or about 40%, at least about or about 45%, at leastabout or about 50%, at least about or about 55%, at least about or about60%, at least about or about 65%, at least about or about 66%, at leastabout or about 67%, at least about or about 68%, at least about or about69%, at least about or about 70%, at least about or about 75%, at leastabout or about 80%, at least about or about 90%, at least about or about95%, at least about or about 100%, at least about or about 150%, atleast about or about 200%, at least about or about 250%, at least aboutor about 300%, at least about or about 400%, at least about or about500%, or a higher percentage more, compared to neutrophil recovery in asubject that is not administered the pre-treated HSCs; or theMYC-composition, the Bcl-2-composition, or both.

Enhanced Hematopoietic Compartment Autoreconstitution

In other embodiments, administering a MYC-composition of the presentdisclosure to a subject in need of autoreconstitution enhanceshematopoietic compartment autoreconstitution in the subject. Forexample, administering the MYC-composition may improve the rate ofautoreconstitution, increase the amount of hematopoietic compartmentcells that are formed, or reduce loss of hematopoietic compartment cellsin the subject, as compared to a subject that is not administered theMYC-composition.

Administration of a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both may enhancehematopoietic compartment autoreconstitution by at least about or about25% to art least about or about 500%, for example, at least about orabout 25%, at least about or about 30%, at least about or about 31%, atleast about or about 32%, at least about or about 33%, at least about orabout 34%, at least about or about 35%, at least about or about 40%, atleast about or about 45%, at least about or about 50%, at least about orabout 55%, at least about or about 60%, at least about or about 65%, atleast about or about 66%, at least about or about 67%, at least about orabout 68%, at least about or about 69%, at least about or about 70%, atleast about or about 75%, at least about or about 80%, at least about orabout 90%, at least about or about 95%, at least about or about 100%, atleast about or about 150%, at least about or about 200%, at least aboutor about 250%, at least about or about 300%, at least about or about400%, at least about or about 500%, or more, compared to hematopoieticcompartment autoreconstitution in a subject that is not administered theMYC-composition composition, the Bcl-2-composition, or both.

As disclosed herein, hematopoietic compartment autoreconstitutionincludes, without limitation, T-cell compartment autoreconstitution,B-cell compartment autoreconstitution, NKT-cell autoreconstitution, andNK-cell compartment autoreconstitution; myeloid cell compartmentautoreconstitution, such as monocyte autoreconstitution, macrophageautoreconstitution, basophil autoreconstitution, eosinophilautoreconstitution, erythrocyte autoreconstitution, megakaryocyteautoreconstitution, platelet autoreconstitution, and dendritic cellautoreconstitution; and neutrophil recovery.

Composition Formulations

Certain aspects of the present disclosure relate to compositionscontaining a MYC-composition of the present disclosure, aBcl-2-composition of the present disclosure, or both for pre-treatingHSCs of the present disclosure, and for treating subjects in need of anHSC transplant. Other aspects relate to a first composition containingHSCs for achieving hematopoietic compartment reconstitution in a subjectin need thereof, and a second composition containing a MYC-compositionof the present disclosure, a Bcl-2-composition of the presentdisclosure, or both for enhancing hematopoietic compartmentreconstitution in the subject. Other aspects of the present disclosurerelate to a composition containing a MYC-composition of the presentdisclosure, a Bcl-2-composition of the present disclosure, or both forenhancing hematopoietic compartment autoreconstitution in a subject inneed thereof.

In some embodiments, compositions of the present disclosure areformulated in a conventional manner using one or more physiologicallyacceptable carriers including, e.g., excipients and auxiliaries whichfacilitate processing of the active compounds into preparations whichare suitable for pharmaceutical use. Suitable pharmaceuticallyacceptable carriers include, without limitation, saline, aqueous buffersolutions, solvents and/or dispersion media. The use of such carriers iswell known in the art. The carrier is preferably sterile. In someembodiments, the carrier is stable under the conditions of manufactureand storage and preserved against the contaminating action ofmicroorganisms, such as bacteria and fungi, through the use of, forexample and without limitation, parabens, chlorobutanol, phenol,ascorbic acid, or thimerosal.

Examples of materials and solutions that can serve as pharmaceuticallyacceptable carriers include, without limitation: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

Proper formulation of the compositions of the present disclosure may bedependent upon the route of administration chosen. A summary ofpharmaceutical compositions described herein is found, for example, inRemington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton,Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975;Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms,Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms andDrug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999).

The compositions of the present disclosure can facilitate administrationof a compound of the present disclosure (e.g., HSCs or a MYC-compositionor a Bcl-2-composition) to a subject or cell. In certain embodiments ofthe methods of the present disclosure, therapeutically effective amountsof compounds described herein are administered in a pharmaceuticalcomposition to a subject having a disorder, disease, or condition to betreated. In some embodiments, the subject is a human patient. In otherembodiments, the subject is a non-human animal, including withoutlimitation, a dog, cat, horse, cow, pig, sheep, goat, chicken, monkey,rat, mouse, or the like.

The HSC compositions, MYC-composition-containing compositions, and/orBcl-2-composition-containing compositions of the present disclosure maybe utilized either alone or in combination with one or more additionaltherapeutic agents. For example, cytokines, growth factors, antibodies,and/or small molecule modifiers that are known to aid in reducing thetime required to reconstitute mature hematopoietic lineages after HSCtransplantation, may administered in combination with the HSC,MYC-composition-containing, and/or Bcl-2-composition-containingcompositions. For example, monoclonal antibodies and/or small moleculemodifiers that target enzymes affecting E-selectin expression canimprove HSC homing to bone marrow niches after an HSC transplantation.Other examples include, without limitation, antibodies or smallmolecules that affect adhesion proteins such and β6 integrin, G-MCSF,G-CSF is the same, and Epo. Accordingly, certain embodiments of themethods of the present disclosure further include administering a thirdcomposition containing at least one cytokine, growth factor, antibody,and/or small molecule modifier. In some embodiments, the cytokine and/orgrowth factor composition further contains a pharmaceutically acceptablecarrier.

The compositions of the present disclosure may be administered to asubject in any suitable manner, including, without limitation, one ormore of multiple administration routes, such as, oral, parenteral (e.g.,intravenous, subcutaneous, intramuscular), intranasal, buccal, rectal,or transdermal administration routes.

Composition formulations of the present disclosure include, withoutlimitation, aqueous liquid dispersions, oil emulsions, self-emulsifyingdispersions, solid solutions, liposomal dispersions, aerosols, soliddosage forms, powders, immediate release formulations, controlledrelease formulations, fast melt formulations, tablets, capsules, pills,delayed release formulations, extended release formulations, pulsatilerelease formulations, multiparticulate formulations, and mixed immediateand controlled release formulations.

Pharmaceutical compositions of the present disclosure (e.g., a PTD-MYCcomposition, a PTD-Bcl-2 composition, or a cytokine and/or growth factorcomposition) are optionally manufactured in a conventional manner,including, without limitation, conventional mixing, dissolving,granulating, dragee-making, levigating, emulsifying, encapsulating,entrapping or compression processes.

In some embodiments, pharmaceutical compositions of the presentdisclosure (e.g., a PTD-MYC composition, a PTD-Bcl-2 composition, or acytokine and/or growth factor composition) are in unit dosage formssuitable for single administration of precise dosages. In unit dosageform, the formulation is divided into unit doses containing appropriatequantities of one or more compound. In some embodiments, the unit dosageis in the form of a package containing discrete quantities of theformulation. Non-limiting examples include packaged tablets or capsules,and powders in vials or ampoules. Aqueous suspension compositions areoptionally packaged in single-dose non-reclosable containers. In someembodiments, multiple-dose re-closeable containers are used. In certainembodiments, multiple dose containers contain a preservative in thecomposition. Formulations for parenteral injection may be presented inunit dosage forms, which include, without limitation, ampoules or inmulti-dose containers with an added preservative.

The following Examples are merely illustrative and are not meant tolimit any aspects of the present disclosure in any way.

EXAMPLES Example 1: Accelerated Hematopoietic Reconstitution In Mice

The following example describes the results of treating mice with aTAT-MYC fusion protein after transplantation with expanded bone marrowcells (e.g. protein transduced longterm hematopoietic stem cells(ptlt-HSCs)).

Materials and Methods

Cohorts of 4-6 week old female C57BL/6J mice were obtained from JacksonLaboratories (Bar Harbor, Me.). The mice were treated with 5 mg/mouse of5-fluorouracil (5FU), intravenously. Bone marrow (BM) cells werecollected from the tibia and femur bones 5 days after 5FU treatment. Thered blood cells were lysed by incubation in 5 ml sterile TAC buffer (135mM NH4CL, 17 mM Tris Ph 7.65). The bone marrow cells were expanded in BMMedium (DMEM containing 15% FCS, 100 units per ml Penn/Strep, MEM NEAA(Gibco), 10 mM HEPES, recombinant murine IL-3, IL-6, and SCF)supplemented with 5 μ/ml recombinant Tat-Myc, and 10 μ/ml recombinantTat-Bcl-2. Cells were cultured for 21 days with a BM medium change every48 hr to refresh the Tat-fusion proteins.

Cytokines were prepared by plating 293FT cells in 150 mm plates at12×10⁶ cells per plate in D10 media (DMEM, 10% FBS, 100 units per mlPenn/Strep, MEM NEAA (Gibco), 2 mM L-glutamine (Gibco)). The cells weretransfected with 30 μg total DNA per plate consisting of 10 μgpcDNA3.1-SCF, 10 μg pcDNA3.1-IL3, and 10 μg pcDNA3.1-IL6 or 10 μgpcDNA3.1-TPO, 10 μg pcDNA3.1-Flt3-L, and 10 μg pcDNA3.1-GM-CSF usingcalcium phosphate (Young, R. M., et al. (2008). Future Oncology, 4,591-4.). The following day, the media was removed and was replaced with100 ml D10 media. Cells were incubated at 37° C./5% CO2 for 4-5 days.The media was collected, sterile filtered, and frozen at −20° C. in 30ml aliquots.

After 21 days, 5×10³ expanded bone marrow cells were transplanted intosublethally irradiated Rag-1^(−/−) mice on a C57/BL6 background (JacksonLaboratory) that received 350 Rads of radiation just prior to injectionof the BM cells via the tail vein. The expanded cells were washed 3times in PBS prior to injection via the tail vein in 200 μl PBS.

After 24 hours, one cohort of mice was injected intramuscularly with 10μg TAT-MYC emulsified in corn oil. The emulsion is prepared by adding 30μg Tat-MYC into lml of corn oil. Just prior to injection, the corn oilcontaining the Tat-MYC is passed several times from 1 syringe to asecond syringe through a 2-way stopcock. 300 ul of the emulsioncontaining 10 μg of Tat-MYC is injected IM into the mouse, just to theside of the tail.

Reconstitution of the lymphoid compartment was monitored by flowcytometric analysis (FACS) of peripheral blood samples obtained byvenipuncture of the tail at 4 weeks and 8 weeks following transplant.Peripheral blood mononuclear cells (PBMCs) were monitored by FACS forTCRβ and for B220 expression.

Results

As shown in FIGS. 1 and 3, accelerated development of T-cells was seenin mice treated with TAT-MYC following bone marrow transplant with exvivo expanded bone marrow cells. Cohorts of Rag-1^(−/−) mice weresub-lethally irradiated and given transplants of 5×10³ expanded bonemarrow cells (FIGS. 1C and 1D; FIGS. 3C and 3D). Half of the irradiatedand transplanted mice in each cohort were injected with 10 μg TAT-MYC 24hours after the transplant (FIGS. 1D and 3D). FACS analysis of wild-type(untreated and non-irradiated) Rag-1^(−/−) mice (FIGS. 1A, 0.9% TCRβ+cells and 3A, 0.8% TCRβ⁺ cells) and C57BL/6 mice (FIGS. 1B, 34.3% TCRβ⁺cells and 3B, 34.3% TCRβ⁺ cells) are also provided as a control.

Mice were tested for T cell reconstitution at 4 weeks (FIG. 1) and 8weeks (FIG. 3) by FACS analysis of their peripheral blood. FIG. 1D showsthe 4-week levels of T cells in the peripheral blood of mice treatedwith TAT-MYC (5.6% TCRβ⁺ cells), as compared to the control (FIG. 1C)that was not injected with TAT-MYC (0.2% TCRβ⁺ cells). FIG. 3D shows the8-week levels of T cells in the peripheral blood of mice treated withTAT-MYC (13.1% TCRβ⁺ cells), as compared to the control (FIG. 3C) thatwas not injected with TAT-MYC (4.2% TCRβ⁻ cells). At 4 weeks, theTAT-MYC injected mice showed T cell levels similar to those of controlmice not injected with TAT-MYC at 8 weeks. As the average time to T-cellrecovery is 8-10 weeks, this represents an acceleration of about 50-60%.

As shown in FIGS. 2 and 4, accelerated development of B cells was alsoseen in mice treated with TAT-MYC following bone marrow transplant withexpanded bone marrow cells. Cohorts of Rag-1^(−/−) mice on a C57/BL6background were sub-lethally irradiated and given transplants of 5×10³expanded bone marrow cells. Half of the irradiated and transplanted micein each cohort were injected with 10 μg TAT-MYC 24 hours after thetransplant (FIGS. 2D and 4D). FACS analysis of wild-type (untreated andnon-irradiated) Rag-1^(−/−) mice (FIGS. 2A, 1.15% B220⁺ cells and 4A, 1%B220⁺ cells) and C57BL/6 mice are also provided as a control (FIGS. 2B,21.4% B220⁺ cells and 4B, 29.2% B220⁺ cells).

Mice were tested for B cell reconstitution at 4 weeks (FIG. 2) and 8weeks (FIG. 4) by FACS analysis of their peripheral blood. FIG. 2D showsthe 4-week levels of B cells in the peripheral blood of mice treatedwith TAT-MYC (12.1% B220⁺ cells), as compared to the control (FIG. 2C)that was not injected with TAT-MYC (0.3% B220⁺ cells). FIG. 4D shows the8-week levels of B cells in the peripheral blood of mice treated withTAT-MYC (5.4% B220⁺ cells), as compared to the control (FIG. 4C) thatwas not injected with TAT-MYC (1.2% B220⁺ cells). At 4 weeks, theTAT-MYC injected mice showed B cell levels higher than those of controlmice not injected with TAT-MYC at 8 weeks. As the average time to B-cellrecovery is 8-12 weeks, this represents an acceleration of about 50-67%.

Example 2: Accelerated Hematopoietic Reconstitution In Mice

The following example describes the results of treating mice with aTAT-MYC fusion protein following freshly isolated whole bone marrowtransplantation.

Materials and Methods

For whole bone marrow transplantations in mice, donors and recipientswere both on a C57/BL6 background.

Bone marrow cells were flushed from femurs and tibial bones obtainedfrom two donor wild type C57/BL6 mice. The harvested cells weretransferred into D10 complete medium (DMEM supplemented with 10% heatinactivated fetal calf serum, 100 units/ml penicillin/streptomycin, 10μg/ml L-glutamine, as well as MEM NEAA). The bone marrow aspirates weredissociated into single cell suspensions and pelleted by centrifugation.The red blood cells were lysed by incubation of the cell suspension in ahypotonic buffer (135 mM NH₄Cl, 17 mM Tris, pH 7.65). The remainingcells were then washed in D10 medium followed by two washes with PBS andkept cold until transplantation into Rag-1^(−/−) mice the same day.

The recipient Rag-1^(−/−) mice were irradiated with 350 Rads (whole bodyirradiation). Recipient mice were given 1×10⁶ whole bone marrow cellsvia tail vein injection. 24 hours after the bone marrow cell transplant,mice were given 10 μg of TAT-MYC emulsified in 300 μl of corn oil asdescribed in Example 1. The TAT-MYC was delivered via intramuscularinjection.

Reconstitution of the lymphoid compartment was monitored by flowcytometric analysis (FACS) of peripheral blood samples obtained byvenipuncture of the tail. Specifically, samples were monitored for theappearance of CD4 or CD8 expressing T-cells (TCRβ), as well as B-cells(CD19 and B220 expressing cells).

Spleen cell samples from chimeric mice were also measured for theability to respond to mitogenic stimulation. Spleen-derived T-cells andB-cells were labeled with CFSE and activated with either antibodies toCD3 (T-cells), or antibodies to IgM and CD40 (B-cells). The cells wereevaluated for proliferation, as determined by dilution of the CFSEsignal, using FACS, 72 hours after stimulation.

Results

Cohorts of 5 mice each (Rag-1^(−/−) mice) were sub-lethally irradiatedand given transplants of 10⁶ whole BM cells obtained from female C57/BL6donor mice. The transplant recipient mice were then either injected withTAT-MYC 24 hours later or not treated. The chimaeric mice weremaintained in the vivarium for observation for 4 weeks. At that point,the mice were euthanized, and PBMCs and spleens were collected. Thespleens were used to generate single cell suspension. Those cells werethen stained with fluoresceinated antibodies to murine CD4 and CD8, andanalyzed by flow cytometry.

FIGS. 5 and 6 show the accelerated development of T cells in micetreated with TAT-MYC following freshly isolated whole bone marrowtransplant. Cohorts of Rag-1^(−/−) mice on a C57/BL6 background weresub-lethally irradiated and given transplants of 1×10⁶ whole bone marrowcells. Half of the transplanted cohorts were injected with TAT-MYC 24hours after the transplant.

FIG. 5A shows the level of peripheral blood CD4 and CD8 T cells incontrol Rag-1^(−/−) mice that did not receive irradiation, a celltransplant, or treatment with Tat-Myc (0.03% CD4⁺ and 0.8% CD8⁺ cells).FIG. 5B shows the level of peripheral blood CD4 and CD8 T cells inuntreated and non-irradiated wildtype C57BL/6 mice as a control (13.1%CD4⁺ and 11.1% CD8⁺ cells). FIG. 5D shows the detection of both CD4 andCD8 T-cells in the peripheral blood of mice treated with TAT-MYC (14.9%CD4⁺ and 8.6% CD8 cells), as compared to the mice (FIG. 5C) that werenot injected with TAT-MYC (4.2% CD4⁺ and 3.2% CD8 cells). At 4 weeks,mice treated with TAT-MYC 24 hours after whole bone marrow transplanthave approximately the same levels of CD4 and CD8 T cells as wildtypeC57BL/6 mice.

FIG. 6 graphically depicts the percentage of T cells for the full cohortof mice described above and shown in FIG. 5. FIG. 6A shows the percentCD4⁺ cells in the peripheral blood at 4 weeks post-transplant in wildtype C57BL/6 (First column), in Rag-1^(−/−) mice irradiated andtransplanted, but not treated with TAT-MYC (Second column), and inRag-1^(−/−) mice irradiated, transplanted, and injected with 10 μgTAT-MYC (Third column). FIG. 6B shows the percent CD8⁺ cells in theperipheral blood at 4 weeks post-transplant in wild type C57BL/6 (Firstcolumn), in Rag-1^(−/−) mice irradiated and transplanted, but nottreated with TAT-MYC (Second column), and in Rag-1^(−/−) miceirradiated, transplanted, and injected with 10 TAT-MYC (Third column).

FIGS. 7 and 8 show the accelerated development of B-cells in micetreated with TAT-MYC following freshly isolated bone marrow transplant.Cohorts of Rag-1^(−/−) mice on a C57/BL6 background were sub-lethallyirradiated and given transplants of 1×10⁶ whole bone marrow cells. Halfof the transplanted cohorts were injected with TAT-MYC 24 hours afterthe transplant.

FIG. 7A shows the level of peripheral blood CD19×B220 B cells in controlRag-1^(−/−) mice that did not receive irradiation, a cell transplant, ortreatment with TAT-MYC (0.2% CD19⁺×B220⁺ cells). FIG. 7B shows the levelof peripheral blood CD19×B220 B cells in untreated and non-irradiatedwildtype C57BL/6 mice as a control (25.8% CD19⁺×B220⁺ cells). FIG. 7Dshows the detection of both CD19×B220 B cells in the peripheral blood ofmice treated with TAT-MYC (5.2% CD19⁺×B220⁺ cells), as compared to themice (FIG. 7C) that were not injected with TAT-MYC (1.4% CD19⁺×B220⁺cells). At 4 weeks, mice treated with TAT-MYC 24 hours after whole bonemarrow transplant have significantly higher levels of CD19×B220 B cellsas transplanted control mice not treated with TAT-MYC.

FIG. 8 graphically depicts the percentage of B cells for the full cohortof mice described above and shown in FIG. 7. FIG. 8A shows the percentCD19×B220⁺ cells in the peripheral blood at 4 weeks post-transplant inwild type C57BL/6 (First column), in Rag-1^(−/−) mice irradiated andtransplanted, but not treated with TAT-MYC (Second column), and inRag-1^(−/−) mice irradiated, transplanted, and injected with 10 μgTAT-MYC (Third column).

FIG. 9 shows that T-cells and B-cells that developed in chimeric micetransplanted with whole bone marrow and treated with TAT-MYC 24 hoursafter transplant were functional and proliferated following stimulationof their antigen receptors. Spleen-derived T-cells and B-cells werelabeled with CFSE and activated with either antibodies to CD3 (T-cells),or antibodies to IgM and CD40 (B-cells). The cells were evaluated forproliferation, as determined by dilution of the CFSE signal, using FACS,72 hours after stimulation.

As shown in FIG. 9A, 33.3% of the spleen cells from mice giventransplants of 1×10⁶ whole BM cells, but not treated with TAT-MYC,showed T cell blasting upon stimulation with anti-CD3. The spleen cellsfrom the chimeric mouse given transplants of 1×10⁶ whole BM cells andtreated with TAT-MYC showed 36.6% T cells blasting after CD3 stimulation(FIG. 9C). Similarly, FIG. 9B shows 6.92% of the spleen cells from micegiven transplants of 1×10⁶ whole BM cells, but not treated with TAT-MYCshowed B cell blasting upon stimulation with anti-CD40 and anti-IgM. Thespleen cells from the chimeric mouse given transplants of 1×10⁶ whole BMcells and treated with TAT-MYC showed 15.8% B cells blasting after CD40and IgM stimulation (FIG. 9D). These data show that the mature lymphoidcells obtained from HSC chimaeric mice that received TAT-MYC treatmentwere able to blast and undergo cell division following activationthrough their antigen receptors.

Example 3: Induction of Hematopoietic Reconstitution In Mice Treatedwith 5-fluorouracil

The following example describes the results of treating mice with aTAT-MYC fusion protein after administration of 5-fluorouracil (5-FU), achemotherapeutic agent known to be toxic to the hematopoieticcompartment.

Introduction

Current approaches for reversing bone marrow failure that arises fromvarious environmental insults or disease, among others, basically relyon the administration of growth factors and red blood cell transfusionsfor supportive therapy. The ultimate goal of these approaches is toencourage the remaining endogenous HSCs to mobilize and repopulate thehematopoietic compartment (auto-reconstitution). However, the currentapproaches are inefficient and in many cases simply delay therequirement for a myeloablative bone marrow transplant.

In addition, the sensitivity of HSCs to chemotherapeutic drugs as wellas radiation has historically limited the doses of each therapy that canbe applied to a patient with a solid tumor, for example. The loss ofHSCs and hematopoietic compartment are one of the early signs oftherapy-related toxicity in cancer patients. The ability to spare theHSC compartment from the therapeutic agents used for cancer couldsignificantly change the approaches that are currently used to deliversuch treatments.

The above results in Examples 1 and 2 utilizing TAT-MYC in the contextof transplantation of HSCs with a low number of donor cells showed thatTAT-MYC could also be useful in promoting auto-reconstitution of thehematopoietic compartment in patients with bone marrow failure syndrome.Although not intending to be bound by theory, it is believed thattreatment with TAT-MYC would target a small number of the remainingresident HSCs and induce such HSCs to divide and give rise todifferentiated hematopoietic lineages.

Material and Methods

Cohorts of 4-6 week old female C57/BL6 wild type (WT) mice were used forthese experiments. Cohorts of 5 mice were treated intravenously with5-flouorouracil (5 mg/mouse) alone, or followed by a treatment witheither 10 μg/mouse of TAT-MYC or 10 μg/mouse of TAT-Cre (24 hourspost-5FU challenge). Both proteins were emulsified in corn oilimmediately prior to intramuscular injection. In some experiments, micewere pretreated (48 hours in advance of 5FU challenge) with either 10μg/mouse of TAT-MYC or TAT-Cre.

Reconstitution of the lymphoid compartment was monitored by flowcytometric analysis (FACS) of peripheral blood samples obtained byvenipuncture of the tail. Specifically, samples were monitored for theappearance of CD4 or CD8 expressing T-cells (TCRβ), as well as B-cells(CD19 and B220 expressing cells).

Results

Challenge with 5-flourouracil was used both as a model for environmentalinsult, and more broadly as a model for treatment of any bone marrowfailure. FIGS. 10, 11 and 12 show the accelerated auto-reconstitution ofT cells at 2 weeks in mice treated with TAT-MYC 24 hours following5-fluorouracil challenge. Cohorts of C57BL/6 mice were challenged with5FU and either left untreated (FIG. 10A), or injected intramuscularlywith a control protein TAT-CRE (FIG. 10B) or with TAT-MYC 24 hours afterthe 5FU challenge (FIG. 10C).

FIG. 10A shows the level of peripheral blood CD4 and CD8 positive Tcells at 2 weeks in 5FU challenged C57/BL6 mice that did not receivetreatment with TAT-Cre or TAT-MYC following 5FU challenge (3.9% CD4⁺ and2.4% CD8⁺ cells). FIG. 10B shows the level of peripheral blood CD4 andCD8 positive T cells in 5FU challenged C57/BL6 mice that receivedtreatment with 10 μg TAT-Cre following 5FU challenge (5.5% CD4⁺ and2.55% CD8⁺ cells). FIG. 5C shows the level of peripheral blood CD4 andCD8 positive T cells in 5FU challenged C57/BL6 mice that receivedtreatment with 10 μg TAT-MYC following 5FU challenge (14.9% CD4⁺ and9.69% CD8⁻ cells.

FIGS. 11 and 12 graphically depict the percentage of T cells for thefull cohort of mice described above and shown in FIG. 10. FIG. 11 showsthe percent CD4⁺ cells in the peripheral blood at 2 weeks post-5FUchallenge in C57BL/6 mice not treated with either protein (Firstcolumn), in C57BL/6 mice injected with 10 μg TAT-MYC (Second column),and in C57BL/6 mice injected with 10 μg TAT-CRE (Third column). FIG. 12shows the percent CD8⁺ cells in the peripheral blood at 2 weeks post-5FUchallenge in C57BL/6 mice not treated with either protein (Firstcolumn), in C57BL/6 mice injected with 10 μg TAT-MYC (Second column),and in C57BL/6 mice injected with 10 μg TAT-CRE (Third column).

In other experiments, challenge with 5-flourouracil is used as a modelfor protection against environmental insult, including for protectionfor HSCs against side-effects of chemotherapy or radiation therapy.

Cohorts of C57BL/6 mice are left untreated, treated intramuscularly withTAT-MYC, or treated intramuscularly with a control protein (TAT-CRE).Five mg of 5-FU are administered intravenously 24 hours later. Atvarious times after 5FU challenge (optionally Days 3, 5, 7 or more),peripheral blood samples are analyzed by FACS to assess the frequency ofT-cells (CD4⁺ and CD8⁺ T-cells) in the blood. A comparison between micepre-treated with TAT-MYC or the control protein TAT-CRE will indicatewhether TAT-MYC is able to confer chemoprotection to hematopoieticlineages.

Example 4: Induction of Hematopoietic Reconstitution In Mice Treatedwith Genotoxic Stress Agents

In the following example two forms of genotoxic stresses for HSCs,chemical and radiological, are used. Age and gender matched C57/BL6 miceare treated with either TAT-MYC or a control protein, and then subjectedto exposure with either a chemotherapeutic agent such as (5FU) orcyclophosphamide (CTX), or with sub-lethal doses of radiation.Fluctuations in the frequency of mature T-cell and B-cells in theperipheral blood of the mice are monitored, starting 5-7 days afterexposure to the specific stimuli.

Use of TAT-MYC to Confer Chemoprotection to Hematopoietic Lineages invivo

Cohorts of 10 C57/BL6 mice are injected with TAT-MYC, injected with anegative control (e.g. TAT-CRE), or left untreated. 24, 48, or 72 hourslater (optionally any time point between 1 and 72 hours), we will treat5 mice in each cohort with 5 mg/mouse of 5FU or 4 mg/mouse ofcyclophosphamide (CTX). The other 5 mice of the cohort will be left withonly the initial treatment of TAT-MYC, TAT-CRE, or no injection).Peripheral blood is obtained by venipuncture, and the frequency ofmature T-cells and B-cell cells in the blood is analyzed by FACS.

The mice are assessed for reduced changes in the frequency of murineT-cells and B-cells in the peripheral blood of mice treated with TAT-MYCprior to challenge with 5FU, Busulfan A or Cyclophosphamide (CTX), incontrast to the significant decrease in the frequency of mature T-cellsand B-cells observed in all other mice exposed to those chemotherapeuticagents. Alternatively, the mice are assessed for deceased recovery timeto restore the frequency of murine T-cells and B-cells in the peripheralblood of mice treated with TAT-MYC prior to challenge with either 5FU,Busulfan A or CTX, in compared with the length time needed to seerecovery in the frequency of mature T-cells and B-cells observed in allother mice exposed to those chemotherapeutic agents.

Additional studies involve a dose-escalation (or de-escalation) ofeither 5FU, Busulfan A or CTX on mice that are treated with TAT-MYC todetermine the parameters of increases in doses of chemotherapetic agentsthat are enabled by TAT-MYC treatment.

Use of TAT-MYC to Confer Radioprotection to Hematopoietic Lineages invivo

The experimental setup is essentially the same as above. The onlydifference is that the mice are exposed to a range of doses of radiationrather than to chemotherapeutic agents following treatment with TAT-MYC,TAT-CRE, or no. Accordingly, 350 Rads and 600 Rads may be used for theC57/BL6J mice. The peripheral blood is monitored for the fluctuation ofmature lymphoid or myeloid cells in by FACS.

The mice are assessed for reduced changes in the frequency of murineT-cells and B-cells in the peripheral blood of mice treated with TAT-MYCprior to challenge with sub-lethal or lethal radiation, in contrast tothe significant decrease in the frequency of mature T-cells and B-cellsobserved in all other mice exposed to radiation. Alternatively, the miceare assessed for deceased recovery time to restore the frequency ofmurine T-cells and B-cells in the peripheral blood of mice treated withTAT-MYC prior to challenge with radiation, in compared with the lengthtime needed to see recovery in the frequency of mature T-cells andB-cells observed in all other mice exposed to radiation.

Additional studies involve a dose-escalation (or de-escalation) of dosesof radiation on mice that are treated with TAT-MYC to determine theparameters of increases in doses of radiation that are enabled byTAT-MYC treatment, and possible combination of radiation andchemotherapeutic agents.

Example 5: Accelerated Hematopoietic Reconstitution In Mice FollowingDifferent Routes of Administration

The following example describes the results of treating mice with aTAT-MYC fusion protein administered either intramuscularly orintravenously following freshly isolated whole bone marrowtransplantation.

Materials and Methods

Experiments were performed as described in Example 2 accept that TAT-MYCwas provided using different routes of administration.

Briefly, mice donors and recipients were both on a C57BL/6 background.Bone marrow cells were flushed from femurs and tibial bones obtainedfrom two donor wild type C57/BL6 mice. The bone marrow aspirates weredissociated into single cell suspensions, pelleted by centrifugation,and the red blood cells lysed. The remaining cells were then washed andkept cold until transplantation into Rag-1^(−/−) mice the same day.

The recipient Rag-1^(−/−) mice were irradiated with 350 Rads (whole bodyirradiation). Recipient mice were given 1×10⁶ whole bone marrow cellsvia tail vein injection. 24 hours after the bone marrow cell transplant,mice received an intravenous injection of 10 μg of TAT-MYC dissolved in200 μl PBS or intramuscular injection of 10 μg of TAT-MYC emulsified in300 μl of corn oil as described in Example 1.

Reconstitution of the lymphoid compartment was monitored by flowcytometric analysis (FACS) of peripheral blood samples obtained byvenipuncture of the tail. Specifically, samples were monitored for theappearance of CD4 or CD8 expressing T-cells (CD4×TCRβ), as well asB-cells (IgM×CD19 expressing cells).

Results

Cohorts of 5 mice each (Rag-1^(−/−) mice) were sub-lethally irradiatedand given transplants of 10⁶ whole bone marrow cells obtained fromfemale C57/BL6 donor mice. The transplant recipient mice were theneither injected with TAT-MYC 24 hours later or not treated. Thechimaeric mice were maintained in the vivarium for observation for atleast 4 weeks, at which point peripheral blood was obtained byvenipuncture and levels of T and B cells were assessed by FACS.

FIGS. 13 and 14 show the accelerated development of T cells in micetreated with TAT-MYC following freshly isolated whole bone marrowtransplant. Cohorts of Rag-1^(−/−) mice on a C57/BL6 background weresub-lethally irradiated and given transplants of 1×10⁶ whole bone marrowcells (FIGS. 13C, 13D, and 13E). Two-thirds of the transplanted mice inthe cohort received intravenous (FIG. 13D) or intramuscular (FIG. 13E)injection with 10 μg TAT-MYC 24 hours after the transplant. FACSanalysis of wild-type (untreated and non-irradiated) Rag-1^(−/−) mice(FIG. 13A, 0.579% CD4×TCRβ⁺ cells) and C57BL/6 mice (FIG. 13B, 17.5%CD4×TCRβ⁺ cells) are also provided as a control.

FIGS. 13D and 13E show the detection of CD4×TCRβ⁺ T cells in theperipheral blood of mice receiving intravenous TAT-MYC (32.8% CD4×TCRβ⁺cells) or intramuscular TAT-MYC (18.2% CD4×TCRβ⁺ cells), as compared tothe control (FIG. 13C) that was not injected with TAT-MYC (6.39%CD4×TCRβ⁺ cells). TAT-MYC injected by either route resulted in micehaving approximately the same levels of CD4×TCRβ⁺ T cells as wildtypeC57BL/6 mice.

FIG. 14 graphically depicts the percentage of T cells for the fullcohort of mice described above and shown in FIG. 13. The graph shows thepercent CD4⁺ cells in the peripheral blood at 4 weeks post-transplant inwild type Rag-1^(−/−) mice (NT), in Rag-1^(−/−) mice irradiated,transplanted, and receiving an intravenous injection of TAT-MYC (IV),and in Rag-1^(−/−) mice irradiated, transplanted, and receiving anintramuscular injection of TAT-MYC (IM).

FIGS. 15 and 16 show the accelerated development of B cells in micetreated with TAT-MYC following freshly isolated whole bone marrowtransplant. Cohorts of Rag-1^(−/−) mice on a C57/BL6 background weresub-lethally irradiated and given transplants of 1×10⁶ whole bone marrowcells (FIGS. 15C, 15D and 15E). Two-thirds of the transplanted mice inthe cohort received intravenous (FIG. 15D) or intramuscular (FIG. 15E)injection with 10 μg TAT-MYC 24 hours after the transplant. FACSanalysis of wild-type (untreated and non-irradiated) Rag-1^(−/−) mice(FIG. 15A, 0.071% IgM×CD19⁺ cells) and C57BL/6 mice (FIG. 15B, 23.5%IgM×CD19⁻ cells) are also provided as a control.

FIGS. 15D and 15E show the detection of IgM×CD19⁺ B cells in theperipheral blood of mice receiving intravenous TAT-MYC (FIG. 15D, 2.41%IgM×CD19⁺ cells) or intramuscular TAT-MYC (FIG. 15E, 6.53% IgM×CD19⁺cells), as compared to the control (FIG. 15C) that was not injected withTAT-MYC (1.49% IgM×CD19⁺ cells). TAT-MYC injected by either routeresulted in mice having higher levels of IgM×CD19⁺ B cells thanRag-1^(−/−) mice transplanted with whole bone marrow, but not treatedwith TAT-MYC.

FIG. 16 graphically depicts the percentage of B cells for the fullcohort of mice described above and shown in FIG. 15. The graph shows thepercent CD19×B220⁺ cells in the peripheral blood at 4 weekspost-transplant in wild type Rag-1^(−/−) mice (NT), in Rag-1^(−/−) miceirradiated, transplanted, and receiving an intravenous injection ofTAT-MYC (IV), and in Rag-1^(−/−) mice irradiated, transplanted, andreceiving an intramuscular injection of TAT-MYC (IM).

Example 6: Accelerated Hematopoietic Reconstitution In Mice Transplantedwith Human Cord Blood-Derived HSCS Expanded with TAT-MYC and TAT-Bcl-2

The following example describes the results of expanding cord bloodderived HSCs in vitro using TAT-MYC and TAT-Bcl-2 to form proteintransduced longterm hematopoietic stem cells (ptlt-HSCs), prior totransplantation into sublethally irradiated mice.

Material and Methods

Fresh cord blood cells were obtained from samples that were discardedfrom a local cord blood bank. All human cells were de-identified andexempt from IRB oversight. The total cord volume was split into 20 mlaliquots and diluted 1:1 in PBS. Diluted cord blood (20 mls) was gentlyoverlaid on 20 mls of Ficoll-Paque Plus (Amersham Biosciences Cat#17-1440-03). The cells were spun at 900× gravity for 60 min. The buffycoat was removed with a glass pipette and was washed twice with PBS. Thecells were resuspended in FCB media (Iscove's (Gibco) supplemented with10% human plasma, 100 units per ml Penn/Strep, 30 ml of media containingSCF, IL3 and IL6 and 30 mls of medium containing TPO, FLT3-L, and GM-CSFdescribed above in Example 1).

Two FCB expansion cultures were initiated. The first culture containedFCB medium alone while the second contained FCB media supplemented with5 μg/ml recombinant Tat-MYC, and 10 μg/ml recombinant Tat-Bcl-2. Themedium on both cultures was replaced every 3 days over a 14 dayexpansion. The surface phenotype of the in vitro expanded human HSCs wasassessed by FACS analysis using antibodies against the human antigensCD45, CD34 and CD38.

Fetal cord blood cells (FCBs) expanded in FCB media or FCB mediasupplemented with Tat-MYC and Tat-Bcl2 were injected into NOD/SCID/γc−/−mice (NSG) mice (Jackson Laboratory) that received 180 Rads of radiationjust prior to injection. Expanded FCBs were washed 3 times in PBS andinjected via the tail vein in 200 μl PBS. Eight weeks post-transplant,the bone marrow cells were collected from the tibia and femur bones ofthe transplant NSG mice. The red blood cells were lysed by incubation in5 ml sterile TAC buffer (135 mM NH₄CL, 17 mM Tris Ph 7.65) followed by 2washes D10 media. The BM cells were analyzed by flow cytometry usingantibodies to the human antigens CD45, CD34, CD38, CD3, CD19, CD11b andCD33.

The spleen and thymus were collected from a euthanized NSG mice and asingle cell suspension was generated by mechanical dissociation. Thecells were treated with TAC buffer (135 mM NH₄CL, 17 mM Tris Ph 7.65) tolyse the red blood cells.

We functionally tested the human HSCs harvested from the BM of NSG miceby plating them on MethoCult Optimum (StemCell Technologies), andexamined their ability to give rise to BFU-E, CFU-M, CFU-G and CFU-GMcolonies. On the day of the BM harvest, 10,000 BM cells in 300 ul D10were added to 4 mls of MethoCult. The 4 ml containing the BM cells weredivided equally between 2, 30 mM dishes, each containing 5000 cells. Thedishes were incubated at 37° in 5% CO₂ for 14 days. The colonies werecounted and identified based on cell morphology using an invertedmicroscope.

Results

As shown in FIG. 17, xenochimaeric NSG mice generated by transplantingHSC expanded in FCB media supplemented with Tat-MYC and Tat-Bcl2(ptlt-HSCs) showed an increase in BM engraftment. Eight weekspost-transplant the NSG mice injected with 1×10⁶ CD34+/CD38lo cellsexpanded in FCB media alone had 0.03% of their bone marrow compartmentderived from the transplanted cells (FIG. 17A, first panel). NSG miceinjected with 1×10⁶ CD34+/CD38lo cells expanded in FCB mediasupplemented with Tat-MYC and Tat-Bcl2 had 18.2% of their bone marrowcompartment derived from the transplanted cells (FIG. 17A, secondpanel). An NSG mouse transplanted with 5×10⁶ fresh cord blood cells wasused as a control for engraftment (FIG. 17A, third panel), and showed2.7% engraftment with transplanted cells.

Human CD45+ cells from the BM, spleen and thymus of xenochimaeric NSGmice generated by transplanting HSC expanded in FCB media supplementedwith Tat-MYC and Tat-Bcl2 were analyzed. FACS analysis shows that 6.8%of the human CD45+ population in the BM also stained positive for thehematopoietic stem cell marker CD34 (FIG. 17B, first panel). Human CD45+cells from the spleen and thymus of these mice were assessed for the Bcell marker CD19 and the T cell marker CD3. The majority of the humanCD45+ cells from the spleen stained positive for the B cell marker CD19(FIG. 17B, second panel, 69.2%), compared to the T cell marker CD3 (FIG.17B, second panel, 7.7%). The majority of CD45+ population in the thymusstained positive for the CD3 T cell marker (FIG. 17B, third panel,91.3%), compared to the B cell marker CD19 (FIG. 17B, third panel,1.2%).

Human CD45+ CD19+ cells from the spleens of xenochimaeric NSG micetransplanted with ptlt-HSCs were labeled with CFSE, and were activatedwith monoclonal antibodies to human CD40 and IgM. The cells wereanalyzed at 72 hours by flow cytometry for dilution of CFSE. FIG. 17Cshows the proliferation profile of the human B-cells that developed invivo in xenochimaeric NSG mice. These results demonstration that the Bcells derived from the transplanted HSCs that received pretreatment withTat-MYC and Tat-Bcl2 can be activated through their B cell receptor.

Human CD45+, CD34+ CD38lo HSCs from the bone marrow of xenochimaeric NSGmice transplanted with ptlt-HSCs were used to seed in MethoCult Optimumto assess the presence of myeloerythroid stem cells. These cells fromNSG mice transplanted with ptlt-HSCs gave rise to more colonies inMethoCult plates (FIG. 17D, FCB TMTB), as compared to cells from controlNSG mice transplanted with cells expanded in media alone (FIG. 17D,FCB). Although colony formation was observed in conditions selective forerythroid (BFU-E), myeloid (CFU-M), granulocyte (CFU-G), and granulocytemacrophage (CFU-GM) lineages, more myeloid and granulocyte growth wasobserved. In addition, some of the colonies could still be observedfollowing serial replating (FIG. 17E). The number of colonies in bothinstances was significantly higher for NSG mice reconstituted with humancord blood cells cultured for 14 days with Tat-MYC and Tat-Bcl-2 thanfor cells obtained from NSG mice reconstituted with fresh,un-manipulated human cord blood cells.

These results show that the CD45+, CD34+CD3810 HSCs from the bone marrowof engrafted mice are hematopoietic stems cell that are able to giverise to all 4 colony types in MethoCult media. Further, the increasedcolony number observed on plates seeded with bone marrow from NSG miceengrafted with FCBs cultures with Tat-MYC and Tat-Bcl2 is reflective ofa greater number of hematopoietic stems cells residing in the bonemarrow niche.

In addition, a cohort of xenochimaeric mice, engrafted with 10⁶ cordblood cells previously expanded in vitro in a cocktail of cytokinessupplemented with Tat-MYC and Tat-Bcl-2 (black squares), were assessedfor myeloid and lymphoid cell differentiation. The CD45 positivepopulation of bone marrow cells (FIG. 17F) and spleen cells (FIG. 17G)were analyzed for CD11b, CD33, CD3, and CD19 expression. Both myeloidand lymphoid cell differentiation was observed in the bone marrow andspleen of these xenochimaeric mice.

Example 7: Accelerated Hematopoietic Reconstitution In Mice Transplantedwith Human Freshly Isolated Whole Cord Blood and Injected with TAT-MYC

The following example describes the results of treating mice with aTAT-MYC fusion protein after transplantation with fresh human cord bloodcells.

Materials and Methods

Human cord blood cells were obtained, and prepared as described inExample 6. Briefly, the cord blood was separated using Ficoll-hypaqueand centrifugation to obtain the buffy coat fraction. Buffy coat cellswere removed, washed twice, then resuspended in PBS, and kept cold untiltransplanted into mice later the same day.

Prior to injecting NOD/SCID/γ^(−/−) (NOG) mice with FCB cells, the micewere irradiated with 180 Rads (whole body irradiation). Each recipientmouse was then given a transplant consisting of 5×10⁵, 1×10⁶, or 5×10⁶human FCB cells via tail vein injection. 24 hours after the bone marrowcell transplant, mice received an intramuscular injection of 10 μg ofTAT-MYC or 10 μg TAT-CRE emulsified in 300 μl of corn oil.

Engraftment was monitored with flow cytometry to assess for the presenceof human CD45 positive cells in the peripheral blood, bone marrow, andspleen of the xenochimeric mice eight weeks after transplantation.

Results

As shown in FIGS. 18, 19 and 20, accelerated development of human cellswas seen in the peripheral blood (FIG. 18), bone marrow (FIG. 19), andspleen (FIG. 20) of xenochimeric NOD/SCID/βc^(−/−) mice treated withTAT-MYC following xenotransplantation of human fresh fetal cord bloodcells. Cohorts of NOD/SCID/βc^(−/−) mice were given a sub-lethal dose ofradiation followed by 5×10⁵ cord blood cells (FIGS. 18A and 18D; FIGS.19A and 19D; FIGS. 20A and 20D), 1×10⁶ (FIGS. 18B and 18E; FIGS. 19B and19E; FIGS. 20B and 20E), or 5×10⁶ (FIGS. 18C and 18F; FIGS. 19C and 19E;FIGS. 20C and 20E). 24 hours after the transplant, half of the mice ineach cohort were injected with TAT-MYC (FIGS. 18D, 18E, and 18F; FIGS.19D, 19E, and 19F; FIGS. 20D, 20E, and 20F) and the other half wereinjected with a control protein, TAT-CRE (FIGS. 18A, 18B, and 18C; FIGS.19A, 19B, and 19C; FIGS. 20A, 20B, and 20C)

FIG. 18 shows that control, sub-lethally irradiated NOD/SCID/βc^(−/−)mice, treated with TAT-CRE following xenotransplantation with humanwhole cord blood cells, had in their peripheral blood at eight weeks, 0%of CD45⁺ cells after transplantation of 5×10⁵ cells (FIG. 18A), 0.2% ofCD45⁺ cells (FIG. 18B) after transplantation of 1×10⁶ cells, and 0.7% ofCD45⁺ cells (FIG. 18C) after transplantation of 5×10⁶ cells.Sub-lethally irradiated NOD/SCID/βc^(−/−) mice treated with TAT-MYCfollowing xenotransplantation with human whole cord blood cells, had intheir peripheral blood at eight weeks, 0.09% of CD45⁺ cells aftertransplantation of 5×10⁵ cells (FIG. 18D), 0.1% of CD45⁺ cells (FIG.18E) after transplantation of 1×10⁶ cells, and 9.3% of CD45⁺ cells (FIG.18F) after transplantation of 5×10⁶ cells. Accordingly, FIG. 18F showsthe detection of human T-cells (hCD45 positive cells) in the peripheralblood of chimeric mice 8 weeks post-transplant. As the average time toT-cell recovery is 12-20 weeks, this represents an acceleration of about33-60%.

FIG. 19 shows that control, sub-lethally irradiated NOD/SCID/βc^(−/−)mice, treated with TAT-CRE following xenotransplantation with humanwhole cord blood cells, had in their bone marrow at eight weeks, 0.02%of CD45⁺ cells after transplantation of 5×10⁵ cells (FIG. 19A), 7.7% ofCD45⁺ cells (FIG. 19B) after transplantation of 1×10⁶ cells, and 11.3%of CD45⁺ cells (FIG. 19C) after transplantation of 5×10⁶ cells.Sub-lethally irradiated NOD/SCID/βc^(−/−) mice treated with TAT-MYCfollowing xenotransplantation with human whole cord blood cells, had intheir bone marrow at eight weeks, 0.04% of CD45⁺ cells after atransplantation of 5×10⁵ cells (FIG. 19D), 8.6% of CD45⁺ cells (FIG.19E) after a transplantation of 1×10⁶ cells, and 20.7% of CD45⁺ cells(FIG. 19F) after a transplantation of 5×10⁶ cells.

FIG. 20 shows that control, sub-lethally irradiated NOD/SCID/γc^(−/−)mice, treated with TAT-CRE following xenotransplantation with humanwhole cord blood cells, had in their spleen at eight weeks, 0.9% ofCD45⁺ cells after transplantation of 5×10⁵ cells (FIG. 20A), 13.9% ofCD45⁺ cells (FIG. 20B) after transplantation of 1×10⁶ cells, and 27.6%of CD45⁺ cells (FIG. 20C) after transplantation of 5×10⁶ cells.Sub-lethally irradiated NOD/SCID/βc^(−/−) mice treated with TAT-MYCfollowing xenotransplantation with human whole cord blood cells, had intheir spleen at eight weeks, 1.2% of CD45⁺ cells after transplantationof 5×10⁵ cells (FIG. 20D), 4.9% of CD45⁺ cells (FIG. 20E) aftertransplantation of 1×10⁶ cells, and 258% of CD45⁺ cells (FIG. 20F) aftertransplantation of 5×10⁶ cells.

Example 8: Accelerated Hematopoietic Reconstitution In Mice Transplantedwith Human Freshly Isolated Cord Blood Cells Pretreated with TAT-MYC andTAT-Bcl-2

The following example describes the results of pre-treating fresh humancord blood cells with TAT-MYC and TAT-Bcl-2 prior to transplantationinto mice.

Materials and Methods

Human cord blood cells were obtained and prepared as described inExample 6. Briefly, the cord blood was separated using Ficoll-hypaqueand centrifugation to obtain the buffy coat fraction. Buffy coat cellswere removed, washed twice, and then resuspended in PBS.

Prior to injection into mice, the isolated cord blood cells were exposedto 5 μg/ml TAT-MYC and 5 μg/ml TAT-Bcl-2 for one hour. After exposure tothe fusion proteins, the cells were washed twice with PBS, thenresuspended in PBS at 5×10⁶ cells per 200 ul, and kept cold untilinjected into mice.

Prior to injecting NOD/SCID/βc^(−/−) (NOG) mice with the FCB cells, themice were irradiated with 180 Rads (whole body irradiation). Eachrecipient mouse was then given a transplant consisting of 5×10⁶ humanFCB cells in 200 ul PBS via tail vein injection.

Engraftment was monitored with flow cytometry to assess the presence ofCD45 positive cells in the peripheral blood of the xenochimeric mice.The first bleed was done 8 weeks after the FCB transplant.

After eight months, the mice were euthanized and the bone marrow cellswere collected from the tibia and femur bones of the xenochimaeric NSGmice. The spleen and thymus were also harvested, and were made intosingle cell suspensions by pressing the cells through a sterile wiremesh screen. The red blood cells from the BM, spleen and thymus werelysed in 5 ml sterile TAC buffer (135 mM NH₄CL, 17 mM Tris Ph 7.65)followed by 2 washes in D10 media. The BM, spleen cells and thymus cellswere prepared for FACs analysis to assess the presence of human CD45positive cells.

Results

FIGS. 21 and 22 show that pre-treatment of human fresh whole cord bloodcells with TAT-MYC for one hour prior to transplantation inNOD/SCID/γc^(−/−) (NOG) mice resulted in accelerated development ofhuman CD45 cells in the peripheral blood of the mice, as well asincreased longterm persistence of human CD45 cells in the bone marrow,spleen and thymus of the mice. Cohorts of NOD/SCID/γc^(−/−) mice weregiven a sub-lethal dose of radiation followed by 5×10⁶ cord blood cells.For half of the mice in each cohort, the cells were pre-treated with 5μg/ml TAT-MYC and 5 μg/ml TAT-Bcl-2 in FCB media (FIG. 21B; FIGS. 22B,22D, and 22F); for the other half of the mice in the cohort, the cellswere pre-treated in FCB media alone (FIG. 21A; FIGS. 22A, 22C, and 22E).

FIG. 21 shows that control, sub-lethally irradiated NOD/SCID/γc^(−/−)mice, xenotransplanted with human whole cord blood cells pre-treatedwith FCB media alone, had in their peripheral blood at eight weeks,0.89% CD45⁺ cells (FIG. 21A). In contrast, sub-lethally irradiatedNOD/SCID/γc^(−/−) mice, xenotransplanted with human whole cord bloodcells pre-treated with 5 μg/ml TAT-MYC and 5 μg/ml TAT-Bcl-2 in FCBmedia, had in their peripheral blood at eight weeks, 15.7% CD45⁺ cells(FIG. 21B).

FIG. 22 shows that control, sub-lethally irradiated NOD/SCID/γc^(−/−)mice, xenotransplanted with human whole cord blood cells pre-treatedwith FCB media alone, had at eight months in their bone marrow, 0.04%CD45⁻ cells (FIG. 22A), in their spleen 0.03% CD45⁺ cells (FIG. 22C),and in their thymus, 0.5% CD45⁺ cells (FIG. 22E). In contrast,sub-lethally irradiated NOD/SCID/γc^(−/−) mice, xenotransplanted withhuman whole cord blood cells pre-treated with 5 μg/ml TAT-MYC and 5μg/ml TAT-Bcl-2 in FCB media, had at eight months in their bone marrow,0.37% CD45⁺ cells (FIG. 22B), in their spleen 25.2% CD45⁺ cells (FIG.22D), and in their thymus, 5.4% CD45⁺ cells (FIG. 22F).

In other experiments, 5 μg/ml TAT-MYC and 5 μg/ml TAT-Bcl-2 are used topre-treat separate populations of human cord blood cells prior to beingtransplanted into different cohorts of NOG mice. The inventors expectthat separate incubation of cells with TAT-MYC and TAT-Bcl-2 will stillafford better engraftment and reconstitution results thantransplantation of the same cells without pre-treatment.

Example 9: Accelerated Engraftment with Human G-CSF Mobilized PeripheralBlood HSCs Cultured with Tat-Myc and Tat-Bcl-2

G-CSF mobilized cells were received in a 1 ml volume of elutriated bloodfrom 5 patients who underwent G-CSF mobilization for autologous HSCtransplantation. All G-CSF samples were de-identified and no furtheridentifying information is associated with the cells used for thesestudies. The cells were added drop wise to 10 ml of FCB media. The cellswere washed twice in FCB media and treated with 5 μg/ml recombinantTat-Myc and 5 μg/ml recombinant Tat-Bcl-2 in a 10 ml volume. Cells werecultured for 12 days

The cells were expanded in media supplemented with cytokines plusTat-Myc and Tat-Bcl2 for 12 days. On the 12^(th) day half of the cellsreceived an additional treatment with 5 μg/ml Tat-MYC and 5 μg/mlTat-Bcl2. The other half of the cells were left in FCB media alone. Thecells were incubated for 60 minutes in a 37° incubator. The cells werewashed three times in PBS, and were resuspended at 5×10⁶ cells per 200ul. Engraftment was monitored with flow cytometry to assess the presenceof CD45 positive cells in the peripheral blood of the xenochimeric mice.The first bleed was done 8 weeks after the HSC transplant.

FIG. 23A shows a FACS analysis of the CD45⁺ staining of the peripheralblood from control NSG (FIG. 23A), NSG mice transplanted 8 weeks earlierwith either 1×10⁶ expanded G-CSF mobilized HSCs (FIG. 23B) or 1×10⁶expanded G-CSF mobilized HSC treated with Tat-Myc/Tat-Bcl-2 (FIG. 23C).As shown for the peripheral blood, the inventors expect that the BM,spleen and thymus from mice engrafted with G-CSF mobilized cellspretreated with TAT-MYC and TAT-Bcl-2 will afford better engraftment andreconstitution results than transplantation of the same cells withoutpre-treatment.

Example 10: Accelerated Hematopoietic Reconstitution In Mice Treatmentwith TAT-MYC Following Transplantation with Human G-CSF Mobilized CellsPretreated with TAT-MYC and TAT-Bcl-2

The following example describes the results of pre-treating human G-CSFmobilized cells with TAT-MYC and TAT-Bcl-2 prior to transplantation intomice, and then followed by treatment of the mice with TAT-MYC 24 hourslater.

Materials and Methods

G-CSF mobilized cells were received in a 1 ml volume of elutriated bloodfrom 5 patients who underwent G-CSF mobilization for autologous HSCtransplantation. All G-CSF samples were de-identified and no furtheridentifying information was associated with the cells used for thesestudies. The cells were added drop wise to 10 ml of FCB media. The cellswere washed twice in FCB media and treated with 5 μg/ml recombinantTat-Myc and 5 μg/ml recombinant Tat-Bcl-2 in a 10 ml volume. The cellswere cultured for 12 days.

On Day 12, the expanded G-CSF mobilized cells received a secondtreatment of 5 μg/ml TAT-MYC and 5 μg/ml TAT-Bcl2 for hour prior toinjecting into NSG mice. The cells were washed 3 times with PBS and theninjected at 5×10⁶ cell per mouse in 200 μl PBS via the tail vein. Priorto injecting NOD/SCID/γc^(−/−) (NOG) mice with the HSC cells, the micewere irradiated with 180 Rads (whole body irradiation). 24 hours postinjection with the expanded HSCs, the mice received 10 μg of Tat-MYC or10 μg Tat-Cre in corn oil intramuscularly, or no injection. Eight weekslater the mice were bled.

Engraftment was monitored with flow cytometry to assess the presence ofCD45 positive cells in the peripheral blood of the xenochimeric mice.The first bleed was done 8 weeks after the HSC transplant.

After eight weeks, the mice are euthanized and the bone marrow cells arecollected from the tibia and femur bones of the xenochimaeric NSG mice.The spleen and thymus are also harvested, and are made into single cellsuspensions by pressing the cells through a sterile wire mesh screen.The red blood cells from the BM, spleen and thymus are lysed in 5 mlsterile TAC buffer (135 mM NH₄CL, 17 mM Tris Ph 7.65) followed by 2washes in D10 media. The BM, spleen cells and thymus cells are preparedfor FACs analysis to assess the presence of human CD45 positive cells.

Results

The data indicate that injection of TAT-MYC 24 hours aftertransplantation of HSCs pre-treated with TAT-MYC and TAT-Bcl-2 for onehour prior to transplantation in NOD/SCID/γc^(−/−) (NOG) mice did notappear to accelerate development of human CD45 cells in the peripheralblood of the mice, as compared with control mice injected with TAT-CREor with no Tat-fusion protein injection, but also transplanted with HSCspre-treated with TAT-MYC and TAT-Bcl2.

At eight weeks, the data show no difference between mice treated withTAT-MYC and TAT-CRE in the levels of CD45+ cells in the peripheralblood. Further investigation is needed to determine if an effect existsat earlier or later time points, or in the BM, spleen, and thymus. Atthis point, the data indicate that pretreating the cells with TAT-MYCmay work just as well as injecting the mice with TAT-MYC aftertransplant. To date, we have not observed an increased effect bypretreating the cells and also injecting the mice with Tat-MYC after thetransplant of treated cells.

Example 11: Generation of Biologically Active Tat-Myc and Tat-Bcl-2Fusion Proteins

Fusion proteins having the HIV-1 Tat protein transduction domain (PTD)and either the ORF for human Myc, or a truncated form of human Bcl-2,that has been deleted for the unstructured loop domain (Anderson, M., etal. (1999). Prot Expr. Purif. 15, 162-70), were generated. Therecombinant proteins also encoded a V5 peptide tag and a 6-His tag, tofacilitate detection and purification (FIG. 24 and FIG. 25).

pTAT-Myc-V5-6×His (Amp^(R)) and pTAT-Bcl2Δ-V5-6×His(Amp^(R)):

plasmid were generated by PCR amplification of a cDNA encoding humancMyc or human Bcl2 using a forward primer encoding an in frame TATprotein transduction domain of HIV (RKKRRQRRR) (SEQ ID NO: 5). The PCRproducts were cloned into pET101/D-Topo (Invitrogen) vector. Theunstructured loop (A.A. #27-80) was removed from the BCL-2 codingsequence using a Quick Change site directed mutagenesis kit (Stratagene#200521-5).

The proteins were synthesized in E. coli and purified to homogeneity.SDS-PAGE electrophoresis and Coomassie Staining revealed the level ofpurity of the final product used for our studies (FIG. 1B).pTAT-Myc-V5-6×His was transformed into BL21-STAR(DE3) cells (Invitrogen)and protein was induced with 0.5 mM IPTG at 37° C. for 3 hrs. The cellswere lysed in lysis buffer (8 M urea, 100 mM NaH2PO4, 10 mM Tris pH to7.0, 10 mM imidazole, pH 7.2). The lysate was diluted to 6M urea andbrought to 450 mM NaCl, 50 mM NaH₂PO₄, 5 mM Tris pH 7.0. The lysate wastreated with Benzonase (500 units) at room temp for 1 hour, clarified bycentrifugation at 12,000 RPM for 60 min and filtered through a 0.22 μMfilter. Myc-V5-6×His was purified on a nickel affinity column (GE) usinga GE AKTA purifier 10 FPLC. Myc-V5-6×His was refolded by dialyzing intodialysis buffer (450 mM NaCl, 50 mM NaH₂PO₄, 5 mM Tris pH 7.0, 5%glycerol, 1 mM DTT). Endotoxin was reduced by passing the purifiedprotein over an Acticlean Etox column (Sterogen).

Bcl2Δ-V5-6×His protein was induced as described above. The cells werelysed in 50 mL of lysis buffer (200 mM NaCl, 200 mM KCL, 50 mM NaH₂PO₄,5 mM Tris pH 7.0, 5% glycerol, 1 mM DTT) supplemented with 500 unitsBenzonase, 1 mM PMSF, 2 μg/ml Leupeptin, 0.015 units/ml Aprotinin, 5 uMHen Egg Lysozyme (HEL) per 1 L of induced protein, and immediatelyplaced on ice for 1 hour. The cells were sonicated on ice (Dutycycle=50%, Output=5) for 2 sets of 2 minutes. The lysate was cleared bycentrifugation at 12,000 RPM for 60 min and was filtered through a 0.22μM filter. Bcl2Δ-V5-6×His was purified on a nickel affinity column (GE)and endotoxin was removed as described above.

The invention claimed is:
 1. A method of enhancing hematopoieticcompartment reconstitution in a subject suffering from a decrease in theamount of endogenous hematopoietic stem cells comprising: administeringa therapeutically effective amount of a composition comprising aMYC-composition, a Bcl-2-composition, or both, to a subject sufferingfrom a decrease in the amount of endogenous hematopoietic stem cells,wherein the MYC composition and the Bcl-2 composition each comprises aprotein transduction domain (PTD), and wherein hematopoietic compartmentreconstitution is enhanced compared to hematopoietic compartmentreconstitution in a subject that is not administered the composition. 2.The method of claim 1, wherein the decrease in the amount of endogenoushematopoietic stem cells is due to bone marrow failure syndrome.
 3. Themethod of claim 1, wherein the decrease in the amount of endogenoushematopoietic stem cells is due to chemotherapy or radiation therapy. 4.The method of claim 1, wherein the subject has cancer.
 5. The method ofclaim 1, wherein the subject has a hematological malignancy, a myeloma,multiple myeloma, a leukemia, acute lymphoblastic leukemia, chroniclymphocytic leukemia, a lymphoma, indolent lymphoma, non-Hodgkinlymphoma, diffuse B cell lymphoma, follicular lymphoma, mantle celllymphoma, T cell lymphoma, Hodgkin lymphoma, a neuroblastoma, aretinoblastoma, Shwachman Diamond syndrome, a brain tumor, Ewing'sSarcoma, a Desmoplastic small round cell tumor, a relapsed germ celltumor, a hematological disorder, a hemoglobinopathy, an autoimmunedisorder, juvenile idiopathic arthritis, systemic lupus erythematosus,severe combined immunodeficiency, congenital neutropenia with defectivestem cells, severe aplastic anemia, a sickle-cell disease, amyelodysplasia syndrome, chronic granulomatous disease, a metabolicdisorder, Hurler syndrome, Gaucher disease, osteopetrosis, malignantinfantile osteopetrosis, heart disease, HIV, or AIDS.
 6. The method ofclaim 1, wherein the subject has received an organ transplant.
 7. Themethod of claim 1, wherein the subject has received a hematopoietic stemcell (HSC) transplant.
 8. The method of claim 7, wherein the HSCtransplant is autologous HSC transplant or an allogenic HSC transplant.9. The method of claim 1, wherein the subject has received amyeloablative HSC transplantation procedure.
 10. The method of claim 1,wherein the subject has received a non-myeloablative HSC transplantationprocedure.
 11. The method of claim 1, wherein the MYC-composition is aPTD-MYC fusion protein.
 12. The method of claim 11, wherein theMYC-composition is a TAT-MYC fusion protein.
 13. The method of claim 1,further comprising administering a therapeutically effective amount of asecond composition comprising hematopoietic stem cells (HSCs) to achievehematopoietic compartment reconstitution in the subject.
 14. The methodof claim 13, wherein the composition comprising a MYC-composition isadministered before, after, or concurrently with the administering ofthe second composition.
 15. The method of claim 13, wherein thecomposition comprising a MYC-composition is administered at least 1 dayafter the administering of the second composition.
 16. The method ofclaim 13, wherein the second composition comprising HSCs was cultured inthe presence of a MYC-composition, a Bcl-2-composition, or both beforethe administering of the second composition.
 17. The method of claim 13,wherein the second composition comprises conditionally immortalizedhematopoietic stem cells (HSCs).
 18. The method of claim 1, wherein theenhanced hematopoietic compartment autoreconstitution in the subjectresults in enhanced T cell compartment autoreconstitution, enhanced Bcell compartment autoreconstitution, enhanced NK-cell compartmentautoreconstitution, enhanced myeloid cell compartmentautoreconstitution, or neutrophil recovery.
 19. The method of claim 1,wherein hematopoietic compartment reconstitution is accelerated comparedto hematopoietic compartment reconstitution in a subject that is notadministered the composition.
 20. The method of claim 19, whereinadministering the composition comprising a MYC-composition, aBcl-2-composition, or both achieves an at least 50% acceleration inhematopoietic compartment reconstitution compared to hematopoieticcompartment reconstitution in a subject that is not administered thecomposition.